Biochemical and agro-biological diversity of Viburnum opulus genotypes

Central European Journal of Biology

* E-mail: [email protected]

Research Article

Received 24 March 2010; Accepted 22 July 2010

Keywords: Biochemical component • Clone • Cultivar • Genotype • Viburnum opulus

1Kaunas Botanical Garden, Vytautas Magnus University, LT-46324 Kaunas, Lithuania

2Faculty of Natural Sciences, Vytautas Magnus University, LT-44404 Kaunas, Lithuania

3Institute of Horticulture, Lithuanian Centre for Agriculture and Forestry, LT-54333 Babtai, Kaunas reg., Lithuania

Laima Česonienė1,*, Remigijus Daubaras1, Jonė Venclovienė2, Pranas Viškelis3

Biochemical and agro-biological diversity of Viburnum opulus genotypes

Abstract: Interest in the biochemical composition of Viburnum opulus fruit has intensified due to the food industry’s demand for natural vitamins, pigments and other substances that enhance the value of different foods. The present study was conducted to determine the agro-biological and biochemical variability of V.  opulus and to select the genotypes that could best serve as sources of health promoting substances. Twelve selected genotypes were evaluated. ‘Leningradskaya Otbornaya’, V.  opulus var. americanum, ‘Zarnitsa’, and local clone P2 were determined to be the best genotypes for growth in commercial plantations. Fruits of the local clone P3 were characterised by large amounts of total phenolics, ascorbic acid, and reducing sugars. V. opulus var. sargentii and V. opulus var. americanum contained exceptionally large amounts of total phenolics, 1460.0 and 1400.0 mg/100 g,respectively. The amount of ascorbic acid varied from 12.4 to 41.4 mg/100 g, the amount of carotenoids varied from 1.4 to 2.8 mg/100 g, the amount of anthocyanins varied from 23.2 to 44.6 mg/100 g, and the amount of total phenolics varied from 753.0 to 1460.0 mg/100 g. The presence of these large amounts of biologically active compounds enables their use as potent antioxidants. The data describing agro-biological characteristics, biochemical components, and health promoting activities of V.  opulus fruits will increase the understanding of this plant and facilitate its use in the food and pharmaceuticals industry.

1. IntroductionIn recent years, increased attention has been paid to lesser known horticultural plants such as saskatoon, honeysuckle, hardy kiwi, elderberry, and cranberry bush because their fruits are rich with biologically active substances known for their antioxidative properties [1-6]. The genus Viburnum comprises more than 230 species, many of which are used for ornamental purposes, but the species Viburnum opulus L. (European cranberry bush) is known for its bitter, edible fruits as well. V. opulus is widespread in eastern, northeastern, western, and central Europe and in western and eastern Siberia [7]. The American cranberry bush V. opulus L. var. americanum Aiton has been recognised as the same species and is closely similar to V. opulus, which

is native to Europe and northern Asia. V. opulus L. var. sargentii (Koehne) Takeda is native to Korea, Northern China, and Japan [8]. Horticultural selections have been made from plants of both continents, primarily for leaf colour, fruit colour, and growth habit. Prolonged selective pressure on V. opulus has resulted in breeding new cultivars in Russia and Ukraine, these cultivars have large fruit with better gustatory properties and high amounts of biologically active substances.

The species V. opulus has a history of use in food, pharmaceuticals, and medicine. The fruits have been used to treat a wide range of maladies including heart disease, coughs and colds, digestive troubles, and bleeding. In Russia, Ukraine and many Siberian nations the fruits are used an ingredient in fillers, sauces, cakes, and drinks [7,9]. V. opulus fruits are popular in

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Scandinavia, while in Canada they are used as substitutes for cranberries [10]. The use of V. opulus fruits as food ingredients in other countries is less popular due to their bitter taste. As several authors have reported, phenolic acids, anthocyanins, and proanthocyanins have been identified in the fruits of V. opulus var. americanum [11,12]. These compounds confer anti-inflammatory properties, promote blood glucose uptake in diabetics, and improve lipid metabolism. Moreover, the health-promoting aspects of other V. opulus genotypes have also been previously reported [3,13,14] and extracts of dried V. opulus fruit possess antimicrobial properties [15]. The seeds of V. opulus are a good source of antioxidants, especially flavonoids [16,17]. In addition, several studies on biochemical characteristics of the other species, Viburnum dilatatum Thunb, revealed that anthocyanins contribute to antioxidant activities and physiological effects of fruits [18,19].

To increase the use of V. opulus fruit in foods and pharmaceutics, a better understanding of their biochemical components and health-promoting activities is needed; however, currently studies on V. opulus fruit are limited [15]. The aim of this study is to compare the biochemical components of V. opulus accessions to assess important agronomical traits and select the most valuable genotypes as sources of health-promoting components.

2. Experimental Procedures2.1 Plant materialThe accessions of V. opulus, including the Russian cultivars ‘Krasnaya Grozd’, ‘Kijevskaya Sadovaya’, ‘Leningradskaya Otbornaya’, ‘Souzga’, ‘Shukshinskaya’, ‘Zarnitsa’, the Lithuanian cultivar ‘Upninkai’, the local clones P1, P2, and P3, as well V. opulus var. americanum and V. opulus var. sargentii, were selected for evaluation in the field collection of the Kaunas Botanical Garden of VMU. These genotypes were previously selected in an experimental collection according to high yield and disease resistance [20]. The Viburnum genetic resources collection is located in the Central region of Lithuania. The altitude of collection was 76 m above sea level. Ten-year-old V. opulus plants were grown with a 2-m distance between rows and bushes. Soil mineral composition was as follows: 65.0 mg P2O5, 13.0 mg K2O, 11.2 mg Ca, and 3.6 mg Mg per 100 g. The soil was pH 7.02 and a humus content of 10.3 g per 100 g.

2.2 Agro-biological characterisationProductivity and fruiting properties including mean yield per bush, the number of fruit in a raceme, and average

fruit weight were studied. The mean yield per bush was determined by weighing the fruit yield during the plants fully mature stage with measurement of seven bushes being made for each accession. The number of fruit in a raceme was calculated using 20 racemes per bush in three replicates. The average weight of a fruit was measured by using an analytical balance with a sensitivity of 0.01 g (ISHIDA company, Japan, model DJ-150E). The maximum capacity of the balance was 150 g. For each genotype, 50 fruits in three replicates were estimated. V. opulus agro-biological characterisation in this study represent summarized five-year-long (2005-2009) investigations of full-fructiferous bushes.

2.3 Fruit sample preparationTwelve fruit samples from each genotype were prepared for determination of biochemical compounds and antioxidant activities in 2009. Fruits were randomly picked from different bushes of each accession at the mass ripening stage and then mixed. The stone seeds were removed prior to homogenisation. The cranberry bush fruits were homogenised in the dark with a homogeniser (Polytron PT1200E, Kinematica, Switzerland) and were analysed immediately.

2.4 Biochemical evaluationThe titratable acidity (TA) was measured by titrating 10 g of pulp that had been homogenised with 100 ml distilled water. The initial pH of the sample was recorded before titration with 0.1 N NaOH to a final pH 8.2. The acidity was expressed as the percentage of citric acid equivalent to the quantity of NaOH used for the titration. Total soluble solids (TSS) were determined by a digital refractometer (ATAGO PR-32, Atago company, Japan). The dry matter (DM) content was determined by the air oven method after drying at 105°C in a Universal Oven ULE 500 (Memmert GmbH+Co. KG, Schwabach, Germany) to a constant weight [21]. Reducing sugars (RS) were determined by the inversion method and the sucrose content (SC) was determined by reducing sugars before and after inversion [22]. The carotenoid content (CC), expressed as β-carotene, was estimated from the extinction value E1 cm

1% =2592 at 450 nm [23]. The absorbance of the hexane solution was determined at 450 nm against a hexane blank in a Genesys10 UV/VISspectrophotometer (Thermo Spectronic, Rochester, USA). The ascorbic acid content was measured by titration with 2,6-dichlorphenolindophenol sodium salt solution using chloroform for intensely coloured extracts [24].

The amount of total phenolics (TPH) in the fruit extracts was determined with the Folin-Ciocalteu

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reagent according to the method of Slinkard and Singleton (1977) using gallic acid as a standard [25]. The reagent was prepared by diluting a stock solution (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) with distilled water (1:10, v/v). Samples (1 ml in duplicate) were aliquoted into test cuvettes, and 5 ml of Folin-Ciocalteu’s phenol reagent and 4 ml of Na2CO3 (7.5%) were added. The absorbance of all samples was measured at 765 nm using a Genesys10 UV/VIS spectrophotometer (Thermo Spectronic, Rochester, USA) after incubation at 20°C for 1 h. The results were expressed as milligrams of gallic acid equivalent (GAE) per 100 g of fresh weight.

The anthocyanins (TAC) were extracted from 5 g of fresh fruits with methanol acidified with 0.1 M HCl [26]. The fruits were ground with quartz sand, and the extraction was continued with 20 ml aliquots of solvent until the sample became colourless. The extract was diluted with acidified methanol; the absorption was measured on a spectrophotometer Genesys10 UV/VIS (Thermo Spectronic, Rochester, USA) at 530 nm. The anthocyanin content was estimated as cyanidin 3-glucoside at 530 nm using a molar absorption coefficient of 26900 and expressed as milligrams per 100 g of fresh weight [27].

The radical scavenging activity against stable DPPH was determined spectrophotometrically [28]. A stable DPPH radical (C18H12N5O6, 2,2-diphenyl-1-picrylhydrazyl, M = 394.32 g/mol) was purchased from Sigma Aldrich Chemical, Germany and analytical reagent grade methanol was used (Penta, Czech Republic). When DPPH (2 ml) reacts with an antioxidant compound (50 µl) that can donate hydrogen, the DPPH is reduced, resulting in a colour change from from deep-violet to light-yellow. This change was measured every 1 min at 515 nm for 30 min. Radical scavenging activity (RSA) was calculated by the following formula:

RSA=[(AB – AS) / AB] × 100%where AB is the absorption of the blank sample

(t=0 min), and AS is the absorption of the tested sample.

2.5 Statistical analysisThe statistical program package STATIS-TICA6 was used for statistical analysis. Associations between agro-biological characteristics and biochemical compounds were presented as Spearman’s rank correlation coefficients with a sample size of n=36 and 34 degrees of freedom (n-2). Principal component analysis and varimax rotation with Kaiser normalisation were applied. Data were standardized before principal component analysis. Statistical differences were declared at P≤0.05 and 0.01. Quantitative results of chemical examinations were expressed for fresh weight.

3. Results and Discussion3.1 Agro-biological characteristics The long-lasting field evaluations of 35 V. opulus genotypes revealed significant variation in the main agronomical properties. Therefore, productive and diseases resistant accessions were selected for further analysis [29]. The yields of the most productive Russian cultivars, ‘Leningradskaya Otbornaya’, ‘Zarnitsa’, and ‘Krasnaya Grozd’, ranged from 8.5 to 11.7 kg, 4.8 to 13.2 kg, and 4.9 to 12.1 kg respectively in different years of evaluations. As described in the previous study [29], the average yields of the cultivars mentioned above were, 8.5, 6.2, and 5.8 kg respectively per bush. It is of upmost importance to select the most productive V. opulus genotypes to grow to maximise the supply of raw ingredients for putative incorporation in food and medicine. The cultivars ‘Leningradskaya Otbornaya’ and ‘Souzga” had the largest fruits with an average fruit mass of 0.60 g and 0.65 g, respectively. Fruits of the accessions of V. opulus var. sargentii and V. opulus var. americanum had masses of less than 0.50 g. The averaged data describing the main agro-biological characteristics of V. opulus are presented in Table 1.

Table 2 presents the correlations between significant agronomical properties. There was a high positive correlation coefficient of 0.898 (P≤0.01) between the number of racemes per bush and the yield per bush. A low positive correlation (0.296) between fruit weight and yield was determined. There was also a low positive correlation between the amount of fruit per raceme and yield per bush (0.407).

The rotated factor matrix for a two-factor model was created. The factor 1 characterized productivity potential with the strong positive loadings for yield per bush (0.928), amount of fruits per raceme (0.705), and amount of racemes per bush (0.807). It could be defined as a “productivity factor”. On the other hand, the factor 2 was interpreted as a “fruit size factor”. It presented the strong positive loading for fruit weight (0.955), i.e. grouped genotypes in accordance with fruit size.The genotypes were distributed against these two principal factors (Figure 1).

The cultivar ‘Leningradskaya Otbornaya’ and local clone P2, which were characterized by large fruits and good yield were determined to be the best productive genotypes for growing in commercial plantations. V. opulus var. americanum, ‘Zarnitsa’, and ‘Krasnaya Grozd’ were shown to have good productivity and medium fruit size. V. opulus var. sargentii typically had the smallest fruits and medium yield. The cultivar ‘Souzga’ was obviously separated from the other genotypes as it exhibited exceptionally large fruits and

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Characteristic M SD Mmin–Mmax V

Fruit weight, g 0.54 0. 08 0.40-0.65 14.3

Yield, kg/bush 5.07 1.85 2.70-8.49 36.5

Number of fruits per raceme 46.2 14.42 29.6-78.6 31.2

Number of racemes per bush 630.8 269.85 228.0-1039.0 42.8

Amount of AA, mg/100 g 27.7 9.00 12.4-41.4 32.7

Amount of TA, g/100 g 3.2 0.63 1.9-3.9 19.9

Amount of CC, mg/100 g 2.1 0.48 1.4-2.8 22.6

Amount of TSS, g/100 g 11.4 1.08 9.0-13.5 9.4

Amount of DM, g/100 g 16.9 1.45 14.3-19.9 8.6

Amount of RS, g/100 g 7.6 0.82 5.6-8.8 10.9

Amount of SC, mg/100 g 0.85 0.47 0.13-1.9 55.5

TPH, mg/100 g 1106.0 199.64 753.0-1460.0 17.8

TAC, mg/100 g 36.2 6.1 23.2-44.6 16.8

Table 1. Descriptive statistics of Viburnum opulus characteristics.

M – mean; SD – standard deviation; Mmin–Mmax – range of mean values; V – variation coefficient; %.AA – ascorbic acid, TA – titratable acidity, CC – carotenoid content, TSS – total soluble solids, DM – dry matter, RS – reducing sugars, SC – sucrose, TPH – total phenolic content, TAC – total anthocyanins content.

Figure 1. Distribution of V. opulus genotypes with regard to agro-biological characteristics.

Fruit weight Yield per bush Number of fruits per raceme Number of racemes per bush

Fruit weight 1.000 0.296* -0.321** 0.135

Yield per bush 0.296* 1.000 0.407** 0.898**

Number of fruits per raceme -0.321** 0.407** 1.000 0.236

Number of racemes per bush 0.135 0.898** 0.236 1.000

Table 2. Correlation matrix for agro-biological characteristics of V. opulus genotypes.

*, ** significant at P≤0.05 and 0.01, respectively.

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very low productivity. As reported in the previous study [29], the cultivar ‘Souzga’ exhibited lower than average yield per bush and fruit number per raceme. This cultivar could be selected as a potential donor in breeding of new cultivars with well-sized fruits.

3.2 Biochemical assayThe averaged data for different biochemical compounds are presented in Table 1. Medium variation (variation coefficient from 10 to 20%) was determined in TA, RS, TPH, and TAC amounts, and substantial variation in AA (32.7%), CC (22.6%), and SC (55.5) was seen. Variation in TSS and DM were small: 9.4% and 8.6%, respectively. AA is one of the most important water-soluble vitamins, the highest AA amount was detected in the selected Lithuanian clone P3 (41.4 mg/100 g), followed by the genotypes V. opulus var. sargentii and V. opulus var. americanum, which contained 33.5 and 30.5 mg/100 g, respectively. The lowest content of AA was recorded in the Russian cultivars ‘Kijevskaya Sadovaya’ and ‘Leningradskaya Otbornaya’, which had concentrations of 26.0 and 12.4 mg/100 g, respectively. Çam et al. reported an ascorbic acid concentration of 52.7 mg/100 g in V. opulus fruit [16]. Studies of AA in other berries have yielded the following concentrations: blackberry (Rubus fruticosus) 15.5–16.3 mg/100 g [30]; blackcurrant (Ribes nigrum) 150.3-275.8 mg/100 g [31]; American cranberry (Vaccinium macrocarpon Aiton) 11.8-15.8 mg/100 g [32].

Fruits of V. opulus genotypes accumulated on average 2.1 mg/100 g of CC. The CC expressed as β-carotene ranged from 1.4 to 2.8 mg/100 g. The cultivar ‘Leningradskaya Otbornaya’, V. opulus var.

americanum, and V. opulus var. sargentii accumulated the largest amounts of CC: 2.8, 2.0, and 2.1 mg/100 g, respectively.

The differences in total phenolic amounts expressed as GAE were deemed to be caused by genetic variation as all accessions were grown under the same ecological conditions. The average amount of TPH in the fruit of V. opulus was 1106.0 mg/100 g. The TPH content of fresh V. opulus fruit has previously been reported to be 356.6 mg/100 g [16] and 351.3 mg/100 g [33] which is lower than in our study. Fruits of the accessions V. opulus var. sargentii and V. opulus var. americanum contained exceptionally large amounts of TPH: 1460.0 and 1400.0 mg/100 g, respectively. The other cultivars and selected clones of V. opulus demonstrated large variation in TPH amounts, which ranged from 753.0 to 1280.0 mg/100 g. The fruits of other horticultural plants express large amounts of TPH as well. Berries of blackcurrant contain from 498 to 1342 mg/100 g [34]; cranberry, about 315 mg/100 g [35].

The amounts of TAC in V. opulus fruits expressed as cyanidin 3-glucoside ranged from 23.2 to 44.6 mg/100 g. Previous studies have reported a variation of 22.9 to 49.9 mg/100 g of TAC [29] and a variation of 22 to 29 mg/100 g of TAC in V. opulus fruit [36], which is in accordance with our results. The Russian cultivar ‘Leningradskaya Otbornaja’ and the clone P1 had the largest quantities of TAC: 44.6 and 40.4 mg/100 g, respectively. Anthocyanins in V. opulus fruit represented 1.8 to 5.2% of TPH. Anthocyanins comprise in bilberries, red raspberries, and lingonberries 90, 30, and 22%, respectively, of the phenolic profiles [6].

The data of this study corroborate the theory that fruits of different V. opulus genotypes contain different

Table 3. Correlation matrix for biochemical components in fruits of Viburnum opulus genotypes.

AA – ascorbic acid, TA – titratable acidity, CC – carotenoid content, TSS – total soluble solids, DM – dry matter, RS – reducing sugars, SC – sucrose, TPH – total phenolic content, TAC – total anthocyanins content, RSA- radical scavenging activity.*, ** significant at P≤0.05 and 0.01, respectively.

AA TA CC TSS DM RS SC TPH TAC RSA

AA 1.000 -0.227 0.061 0.365* 0.281 -0.210 0.368* 0.735** -0.203 0.373*

TA -0.227 1.000 0.114 0,009 -0.207 0.131 0.181 -0.346* 0.278 0.120

CC 0.061 0.114 1.000 -0.389* -0.440* -0.512** -0.263 0.043 0.344* 0.574**

TSS 0.365* 0,009 -0389* 1.000 0.817** 0.106 0.299 0.409* -0.305 -0.161

DM 0.281 -0.207 -0.440* 0.817** 1.000 0.023 0.098 0.453** -0.474** -0.173

RS -0.210 0.131 -0.512** 0.106 0.023 1.000 0.295 -0.583** -0.244 0.259

SC 0.368* 0.181 -0.263 0.299 0.098 0.295 1.000 0.077 0.121 -0.416*

TPH 0.735** -0.346* 0.043 0.409* 0.453** -0.583** 0.077 1.000 -0.232 -0.141

TAC -0.203 0.278 0.344* -0.305 -0.474** -0.244 0.121 -0.232 1.000 0.109

RSA 0.373* 0.120 0.574** -0.161 -0.173 0.259 0.416 0.141 0.109 1.000

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amounts of biologically active substances. The correlation matrix for biochemical components in fruits of V. opulus accessions is shown in Table 3.

The genotypes expressing large amounts of TPH accumulated more AA as described by a correlation coefficient of 0.735, at P≤0.01. The average amount of CC was 2.1 mg/100 g, which created a moderate negative correlation with reducing sugars; the correlation coefficient was -0.512 (P≤0.01). The TPH was negatively correlated with RS (correlation coefficient of -0.583, at P≤0.01). The genotypes were distributed according to the principal component analysis (Figure 2).

The factor 1 could be characterised as a “biological value factor” with strong positive loadings for TPH (0.835), DM (0.610) and AA (0.831) as well as negative loadings for TAC (-0.694) and TA (-0.787). The factor 2 was interpreted as a “sweetness factor” and was characterised by strong positive loading for RS (0.892) and SC (0.620) but had negative loading for CC (-0.670). Fruits of the local clone P3 were characterised by large amounts of TPH, AA, SC, and RS. This clone separated from the main group with a high positive value. It could be selected as a potential donor for the future breeding of new cultivars with higher amounts of TPH and AA. Genotypes which are characteristic of high TPH and AA amounts should be prospective as raw for the pharmaceutical industry. Fruits of the cultivars ’Souzga’ and ’Zarnitsa’ contained small amounts of TPH, AA, and CC, however these genotypes were found to contain the

largest RS and SC amounts. It could be stated that fruits of the ’Souzga’ and ’Zarnitsa’ cultivars are less astringent, whereas other authors attribute polyphenols to contributors of fruit astringency [10]. Consequently, these cultivars could be the best for applications in food supplements and further breeding of new V. opulus cultivars with more palatable fruits.

The inactivation of DPPH radicals was measured after 15 minutes. During this period the RSA ranged from 52.2 to 56.7%. The strongest RSA in 30 minutes (69.9%) was observed in by the cultivar ‘Krasnaya Grozd’. It has previously been reported that extracts of V. opulus fruit had strong radical scavenging activity [15,16] which supports our findings. The carotenoids and ascorbic acid amounts were positively correlated with RSA (correlation coefficients of 0.574** and 0.373*, respectively). No conclusive relationship between TPH content and antioxidant activity was found in this study (Table 3). Several authors [15,16,30,37] have noted that berry fruits, such as cranberry bush, bilberry, blueberry, chokeberry, blackcurrant, and cranberry are rich sources of antioxidants. Antioxidants inhibit the oxidation of ascorbic acid, carotenoids and unsaturated fatty acids. The results of this study indicate that V. opulus fruit extracts can contribute to the health value of foods because of their RSA. The presence of large amounts of biologically active compounds enables their use as potent antioxidants.

Figure 2. Distribution of V. opulus genotypes with regard to biochemical components.

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4. ConclusionIn recognition of their biochemical value, V. opulus fruits have long been used as natural remedies in the treatment of various diseases, including circulatory, respiratory, digestive, and urinary system ailments. This study corroborates the value of V. opulus as a potential source of biologically active substances for functional food and pharmaceuticals. The examined V. opulus genotypes demonstrated significant biochemical and agro-biological diversity. The data describing the biochemical diversity of V. opulus informed our selection of genotypes with large amounts of phenolics, carotenoids, ascorbic acid, and other health-promoting compounds as the best candidates

for use in food production and pharmaceutical development. Furthermore, V. opulus var. sargentii and V. opulus var. americanum exhibited exceptionally high levels of total phenolics compared to other horticultural plants and the P3 clone was of large amounts of TPH and AA. These V. opulus genotypes could be applied in the production of various pharmaceutical products. Additionally, the genes of cultivar ‘Leningradskaya Otbornaya’ and local clone P2 could be used to breed plants with valuable properties such as high yield and large fruits. By profiling the agro-biological and biochemical properties of different V. opulus plants, these data could enable the breeding of plants with increased fruit yield and substantial health-promoting activity.

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