Bulletin UASMV Agriculture, 66 (1)/2009 Print ISSN 1843-5246; Electronic ISSN 1843-5386

The Morphological Features and UV-VIS Analysis of Some Taxonomic Markers of Genus Viscum

Simona Ioana VICA 1), Dumitrita RUGIN Ă2), Stelian PANTEA 1), Carmen SOCACIU 2)

1)University of Oradea, Faculty of Environmental Protection, 26 Gen. Magheru St., 410048 Oradea; Romania, e-mail: [email protected] 2)University of Agricultural Sciences and Veterinary Medicine, Department of Chemistry and Biochemistry, 3-5 Manastur St., 400372 Cluj-Napoca; Romania

Abstract. European mistletoe ( Viscum album L ., family Santalaceae) is native from Europe and is an evergreen, perennial, hemiparasitic shrub that lives on a wide range of woody plants species. The mistletoe has a number of biological effects, such as anticancer, antimycobacterial, apoptosis – inducing, antiviral, and immunomodulatory activities. In the first part of this study, we present the floral and vegetative characters of five varieties of Viscum album ssp. album that are growing on five different host trees ( Acer campestre, Mallus domestica, Fraxinus excelsior, Populus nigra and Robinia pseudoacacia ). In the second part, we recorded the UV-VIS spectrum of methanolic extract (400 to 700 nm), to determined the level of chlorophyll a and b, and also the ratio of A 280 /A 340 (to identify which the bioactive compounds is predominantly). We find the diference between the stem and leaves from the mistletoe that are growing on the same host, and also between the mistletoe that are growing on different host trees.

Key words : Viscum album , Morphology, chlorophylls, phenolic acid, flavonoid.

INTRODUCTION

Viscum is a genus of about 70-100 species of mistletoes, native from Europe, Africa, Asia, and Australasia (from temperate and tropical regions). Traditionally, the genus Viscum , has been placed in its own family Viscaceae , but recent genetic research by APG II (The Angiosperm Phylogeny Group, 2003) system shows this family to be correctly placed within family Santalaceae (Fig. 1). Different species of Viscum are capable of parasitizing a large number of host species. From a review of literature, Barney et al., 1998, identified 452 host species of Viscum album (96 genera and 44 families). For V. album ssp. album 190 hosts have been identified, for V. album ssp. abieties 10 hosts, and for V. album ssp. austriacum 16 hosts are known (Zuber, 2004). Mbagwu and Onuoha, (2007) present in their studies, the vegetative and floral characters of five Viscum variants from Eastern Nigeria and showed an interesting taxonomic relationships among the investigated variants. This study present the floral and vegetative morphology of Viscum album , that are growing on five different host trees. Also, we recorded the UV-VIS spectrum of methanolic extract (400 to 700 nm), to determined the level of chlorophyll a and b, and also the ratio of A280 /A 340 to determined the level of chlorophyll a and b, and also the ratio of A 280 /A 340 to find a relationship between the balance of phenolic acid and flavonoids.

193 Phylum : Magnoliophyta

Class : Magnoliopsida

Order : Santalales

Family :Loranthacea Family : Opiliaceae Family : Santalaceae

Family : Misodendraceae Family : Olacaceae

Genus: Viscum

Species: Viscum album

Fig. 1. Scientific classification of species Visvum album (according to APG II system)

MATERIALS AND METHOD

Plant material. Different variants of V. album plants were harvested in May 2009, from five different host trees located in North-West of Romania country. They were labeled according with the host trees, thus: Acer campestre (VAJ), Mallus domestica (VAM), Fraxinus excelsior (VAF), Populus nigra (VAP) and Robinia pseudoacacia (VAS) for easy identification. Morphological Studies. The morphological studies were included the vegetative parts and the floral parts. The vegetative parts include the stem, where the stem habit, type, colour and bark were considered, the leaf, where the leaf texture, shape, length and width were also studied. The floral parts, type, colour, shape of fruits, and the seed number were also studied. The length and width of the stem and leaves were measured using a rule. The twenty leaves were measured for each of the variants. The seed number per fruit was obtained by counting the number of seeds in a matured fruits so as to ensure consistency. Photographs of the variants of Viscum album were taken with ordinary camera (Sony, 7.2 Mega Pixels). Spectrophotometric determination of chlorophyll pigments. For extraction of chlorophylls (a and b), 2 g fresh weight of leaves or stem were homogenizated with 5 ml methanol using Ultra Turrax homogenizers. Then,the samples were sonicated for 15 minutes, and centrifugated at 12000 rpm, for 10 minutes. The supernatants were separated and the content of green pigments was determined using JASCO spectrophotometer. The data obtained after the spectrophotometrycal determination, was mathematically processed using formulae proposed by Wellburn, (1994):

Chlorophyll a (µg/g dry matter) = (15.65 A 666 – 7.34 A 653 )

Chlorophyll b (µg/g dry matter) = (27.05 A 653 – 11.21 A 666 )

Where, A x represent the absorbance at x nm.

194

RESULTS AND DISCUSSION

The genus Viscum are woody hemi-parasitic shrubs with branches 15–80 cm long, which grow on different host trees. Mistletoe tend to form a globular shape (Fig. 1, a) which may reach over 1 m in diameter. The foliage is dichotomously or verticillately branching, with opposite pairs of green leaves (Fig 2, c and d) which perform some photosynthesis (minimal in some species, notably V. nudum ), but with the plant drawing its mineral and water needs from the host tree. The leaves are narrowly obovate (Fig. 2, b), approximatelly three or four times as long than broad (Table 1). The colour of leaves are dark green, and present three distinct veins and two less distinct veins running parallel to the leaf margin (Fig. 2, b). In young leaves, the veins are visible on both sides, while in older leaves they are mainly seen on the underside (Büssing, 2000). V. album is dioecious, i.e. part of the plants is female, the other part is male. Flowers are highly reduced and inconspicuous, greenish-yellow colour, and 1–3 mm diameter (Fig. 2, c). The fruit is a berry, white, when mature, containing one seed embedded in very sticky juice. The results of these investigations showed no significant difference relationship among the investigated variants. The only difference, are in the length and width of leaves (Tab. 1). For examples, the biggest leaves are recorder in the case of mistletoe that are growing on R. pseudoacacia (8.40 ± 2.53 cm) and the small leave are recorded in the case of mistletoe that are growing on M. domestica (4.14 ± 1.16). The similar result we obtained to measure of the lenghth of stem branched I and II (Fig. 2,d). The length to breadth index has been used to classify subspecies but the variation is too big to make a clear cut classification (Büssing, 2000). The size of the leaves depends very much from the nutrition of the host tree and the position of the parasite within the host tree. Also, the leaf area may vary for the subspecies (Büssing, 2000). The slight variations could be due to environmental influences and the nutrient of the host plant (Nwachukwu and Mbagwu, 2006). Tab. 1 The dimension of leaves and stems from Viscum album that are growing on five different trees

Dimension (cm) VAJ VAM VAF VAP VAS Length A* 4.77 ± 0.75 8.30 ± 1.30 7.05 ± 0.87 6.83 ± 2.74 7.90 ± 0.93 Length B* 3.80 ± 0.86 5.46 ± 2.18 5.68 ± 1.35 9.36 ± 1.22 8.10 ± 2.78 LEAF Length 4.66 ± 1.12 4.14 ± 1.16 5.08 ± 1.17 7.23 ± 1.27 8.40 ± 2.53 Width 1,23 ± 0,12 1,37 ± 0,15 1,40 ± 0,21 1,80 ± 0,09 2,10 ± 0,32 Ratio 3.78:1 3.02:1 3.62:1 4.01:1 4.00:1 (Length/ Width) * Length A means the dimension of stem branched II, and the length B means the dimension of stem branched I (Fig. 2, d)

Viscum album is a hemi-parasitic plant that depends for water and mineral nutrition on its host but is able to produce carbohydrates by photosynthesis. It contains all pigments, chlorophyll a and b as well as carotenoids thar are necessary for photosynthesis (Luther and Becker, 1987). The site of photosynthesis in plants is predominantly the green leaf. The chlorophylls of higher plants consist of chlorophyll a as the major pigment and chlorophyll b as an accesory pigments. Chlorophyll content as well as chlorophyll ratio can be modified by both internals factors and the environmental conditions. Plant parts other than the leaf that can

195 retain or develop chlorophyll, such as stems, branches, floral parts, fruits and some aerial roots do undergo photosynthesis.

a b

Length B c Length A e

d

Fig. 2. Viscum album morphology. a. Image of Viscum album growing on poplar ( Populus nigra ); b. Aspect of leave; c. The flower of mistletoe; d. Stem branched; e. Adult berry containing one seed

Tab. 2 contains the levels of chlorophyll a and b concentrations (µg/g dry matter) from leaves and stems of Viscum album that are growing on five different host trees, and Fig. 3 shows the comparative absorbance spectrum from 400 to 800 nm of leaves and stems from mistletoe methanol extract. Chlorophylls exhibit two major light absorption bands, one of the blue side of the visible spectrum (< 460 nm) and one in the red (630 -670 nm). The absorbance maxima for chlorophyll a is 666 nm and 434 nm, and for chlorophyll b is 653 nm and 453 nm. The primary difference in pigments composition of mistletoe extracts was the low total chlorophyll (a + b) contents observed in the leaves, comparative with stems, except the leaves of VAM (39.08 µg/g dry matter in leaves and 34.45 µg/g dry matter in stems) and VAS (21.72 µg/g dry matter in leaves and 21.19 µg/g dry matter in stems). Another difference in green pigments was the increase of content of chlorophyll b comparative with a, and this lead to lower ratio chlorophyll a/b. The lowest ratio was recorded in the case of VAF extract from leaves (0.32). Literature data show chlorophyll a/b ratios ranging from 1.8 to 2.7 in different species (Wahid and Rasul, 2005).

196 Tab. 2 The content of chlorophyll pigments (a and b) (µg/g dry matter)from leaves and stem of mistletoe extract

Sample Chlorophyll a Chlorophyll b Total Chlorophyll Ratio (µg/g dry matter) (µg/g dry matter) (a + b) Chlorophyll a / b Leaves VAF 2.93 ± 3.37 19.00 ± 9.86 21.93 0.32 VAP 5.27 ± 3.84 9.27 ± 7.60 14.54 0.57 VAS 8.85 ± 0.36 12.87 ± 1.03 21.72 0.69 VAM 14.58 ± 1.89 24.50 ± 3.10 39.08 0.60 VAJ 7.20 ± 0.59 18.54 ± 2.14 25.74 0.39 Stem VAF 23.37± 6.42 25.67 ± 6.95 49.04 0.91 VAP 12.78 ± 0.77 13.28 ± 1.42 26.06 0.96 VAS 10.11 ± 2.22 11.08± 2.42 21.19 0.91 VAM 16.79 ± 3.65 17.66 ± 1.95 34.45 0.95 VAJ 9.75± 12.18 29.35 ± 4.5 55.06 0.33

1.0

2.5 VAP sample VAF sample 2.0

Leaves 1.5 0.5 Stem Leaves Stem 1.0 Absorbance (A.U.) Absorbance Absorbance (A.U.) Absorbance

0.5

0.0 0.0 400 450 500 550 600 650 700 400 450 500 550 600 650 700 Wavelenght (nm) Wavelenght (nm)

1.5 1.5

VAS sample VAM sample

1.0 1.0

Leaves Leaves Stem Stem

Absorbance (A.U.) Absorbance 0.5 (A.U.) Absorbance 0.5

0.0 0.0 400 450 500 550 600 650 700 400 450 500 550 600 650 700 Wavelenght (nm) Wavelenght (nm)

1.5 VAJ sample

1.0

Leaves Stem

Absorbance (A.U.) 0.5

0.0 400 450 500 550 600 650 700 Wavelenght (nm)

Fig. 3. The spectrum of V. album extract of leaves and stem

197 Fig.4 (a and b) shown the comparative absorption spectra (400 to 800 nm) of leaves and stem from Viscum album (methanolic extract).

1.4 a 2.4 b

2.1 1.2

1.8 1.0 VAF VAF VAJ VAJ 1.5 VAM 0.8 VAM VAP VAP 1.2 VAS VAS 0.6 0.9 Absorbance(a.u.) Absorbance(a.U.) 0.4 0.6

0.2 0.3

0.0 0.0 400 450 500 550 600 650 700 400 450 500 550 600 650 700 Wavelenght (nm) Wavelenght (nm)

Fig.4. Absorption spectra of Viscum album (methanol extract, 10x dilution) from leaves (a) and stems (b).

Recently, great attention has been focused on the role of the antioxidative defense system in oxidative stress. The antioxidants from the medicinal plants may play an important role, protecting the biological functions of cells. In the last years, many research papers are focused on the antioxidant activity of parasitic plants, specially on Viscum coloratum , that has commonly been used for Chinese medicine as a curative for a number of diseases. Shi et al., 2006, have recently found that some compositions of Viscum coloratum can act effectively in vitro as antioxidants and peroxyl radical scavengers. Leu et al., 2006, have isolated and characterized structures of new compounds from V. coloratum , among them, viscolin (1,3- diphenylpropane) and one new flavanone, (2S)-7,4’-dihydroxy-5,3’-dimethoxyflavanone. Tab. 3 contains the absorbance levels registered at 280 nm (specific to phenolic acids) and 340 nm (specific to flavonoids), per g dry matter. The ratio A280 /A 340 ranged between 1.28 (in the case of VAS leaves) and 2.58 (in the case of VAP leaves). In generally, the ratio is higher in leaves comparative with stem, except the VAS and VAJ extracts. Flavones and flavonols have an absorption maximum in the UV region. As protective pigments, they shield plants from the damaging effect of UV light. The irradiation of leaves with UV light induces a strong increase in flavonoid biosynthesis. According to this, the decrease of ratio A280 /A 340 from mistletoe, can be use like as bio- indicators of enhanced UV radiation, especially in late autumn to spring. Tab. 3 The wavelength at 280 and 340 nm of methanolic extract of V. album (dilution 1 x 100)

Sample A280 nm/ dry matter A340 nm/ dry matter A280 / A 340 VAF 2.05 0.97 2.10 VAP 1.90 0.74 2.58 Leaves VAS 1.24 0.97 1.28

VAM 1.93 0.97 2.00 VAJ 1.85 0.93 2.00

VAF 2.24 1.60 1.41 VAP 0.88 0.42 2.08 Stems VAS 0.81 0.43 1.87

VAM 0.91 0.45 2.00

VAJ 1.40 0.68 2.05

198 Few literature data were reported regarding to the antioxidant effects of European mistletoe. At the moment, there are some research studies (Onay-Ucar et al., 2006; Vica ş et al., 2007;Vica ş et al., 2008) regarding the antioxidant activity of methanolic and aqueous extract of mistletoe ( Viscum album ). The authors showed that the antioxidant activity of mistletoe extract depends both the harvesting time and the host trees. Becker and Exner (1980) investigated the flavonoid patterns of Viscum album subspecies from various hosts. They identified quercetin and a series of quercetin methyl ethers, which may be assumed to be accumulated on the plant surface. The major flavonoid constituents of Viscum epicuticular waxes are methyl ethers of quercetin (Haas et al., 2003). Also, the kaempferol ant its methyl ethers, and naringenin were identified in the genus Viscum . At the moment, the phytochemical investigation of parasitic plant belongs to the Viscum genus, didn’t reveal a taxonomic marker from flavonoid class. Haas et al., 2003, suggest that the flavonoid profiles expressed in Viscum epicuticular waxes are not as characteristic as to be of taxonomic value. The taxonomic marker of the Loranthaceae family was quercitrin (Quercitin-3-O- Rhamnoside), already isolated from three phyllogenetically linked genera of Scurrula , Taxillus , Dendrophtoe and Helixanthera (Lohézic-Le-Dévéhat et al., 2002). 4’’-O- acetylquercitrin was the first isolation from Loranthacea family by the Lohézic-Le-Dévéhat et al., (2002), and it will be a specific marker of Scurrula genus. Dendrophthoe falcate , it is a a hemiparasite plant that is used in folklore medicine and belongs to the Loranthaceae family. Different flavonoids were isolated from D. falcate that are growing on six different host plants. Quercitrin was found to be the major common constituent, and the Pattanayak et al., 2008, consider that this flavonoid is a taxonomic marker of this species.

CONCLUSION

The slight morphological differences observed among the V. album varieties could be due to environmental influences and to the host plant nutrition, and not on their genetic characteristics. The composition and contents of photosynthetic pigments appear to be important for taxonomic classification as well as for the determination of physiological characteristics of different varieties of V. album . Therefore, further research on the flavonoid chemistry of these types of mistletoes will be necessary, these secondary metabolites may prove to be useful taxonomic markers.

Acknowledgements. This work was supported by Exploratory Research Projects, ID_696, of The National Authority for Scientific Research, Ministry of Education, Research and Youth, Romania. REFERENCES

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