The main ingredients of the *Viscum album* extract are its three ribosome inactivating proteins or lectins (mistletoe lectins, ML) ML-1, ML-2, and ML-3 (Hajtó et al., 2005), the glycoprotein binding with D-galactose and N-acetyl-D-galactosamine, viscotoxins (VT) (Urech et al., 2006), as well as, oligo- and polysaccharides, alkaloids (Khwaja et al., 1980). The flavonoid patterns of *V. album* from various hosts were investigate by Becker and Exner (1980). They identified quercetin and a series of quercetin methyl ethers, which may be assumed to be accumulated on the plant surface. The epicuticular material of the *V. album* contains preferably the flavonol quercetin and its methyl derivatives, occasionally also the flavonol kaempferol and some of its methyl derivatives, and rarely the flavanone naringenin (Haas et

It has been suggested that pharmacologically active compounds may pass from the host

In recent years, the research studies were focused on the antioxidant activity of mistletoe (Onay-Ucar et al. 2006; Leu et al., 2006; Yao et al., 2006; Shi et. al., 2006; Vicas et al., 2009b;

Many plant extracts exhibit efficient antioxidant properties due their phytoconstituents, such as phenolics, especially phenolic acids and flavonoids (Aqil *et al.,* 2006; Miliauskas *et*

To evaluate the antioxidant capacities of plant extracts, numerous *in vitro* methods have been developed. DPPH (2,2-diphenyl-1-picrylhydrazyl), ORAC (oxygen radical absorbance capacity), Trolox equivalent antioxidant capacity (TEAC), and ferric-reducing ability (FRAP) are among the more popular methods that have been used (Wu *et al.,* 2004). The advantages and disadvantages of these methods have been fully discussed in several

viscotoxins alone (Eggenschwiler et al., 2007).

therapie.de/- Mistletoe in cancer treatment).

al., 2003).

Choudhary et al., 2010).

the host trees to the parasitic plants (Büssing and Schietzel., 1999).

trees to the parasitic plants (Büssing and Schietzel., 1999).

*al.,* 2004) and carotenoids (Stahl and Sies, 2003).

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).

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. 2, 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. The color 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 color, and 1–3 mm diameter (Fig. 2, c). The fruit is a berry, white, when mature, containing one seed embedded in very sticky juice (Fig. 2, e).

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 (Vicas et al., 2009a).

In recent years, antioxidants derived from natural resources, mainly from plants, have been intensively used to prevent oxidative damages. Natural antioxidants have also some advantages over synthetic ones, being obtained easily and economically and have slight or negligible side effects.

Phytochemicals as Nutraceuticals 116 – Global Approaches to Their Role in Nutrition and Health

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,

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. 2, 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. The color 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 color, and 1–3 mm diameter (Fig. 2, c). The fruit is a berry, white, when mature, containing one seed

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

c d e

a b

In recent years, antioxidants derived from natural resources, mainly from plants, have been intensively used to prevent oxidative damages. Natural antioxidants have also some advantages over synthetic ones, being obtained easily and economically and have slight or

2004).

embedded in very sticky juice (Fig. 2, e).

containing one seed (Vicas et al., 2009a).

negligible side effects.

Aqueous extracts of the European mistletoe have been widely used for decades as alternative treatment and adjuvant cancer therapy, particularly in Germany, Austria and Switzerland. The European mistletoe extracts are used in an adjuvant cancer therapy because of their immunostimulatory and simultaneously cytotoxic properties. These effects are usually more evidence for the whole extracts than for purified mistletoe lectins and viscotoxins alone (Eggenschwiler et al., 2007).

Different mistletoe preparations are available for the treatment of cancer. Abnobaviscum®, Helixor, Iscador®, Iscucin® and Isorel® are produced according to anthroposophical methods; other mistletoe preparations include Cefalektin®, Eurixor® and Lektinol®. Iscador was first proposed for the treatment of cancer in 1920, by Rudolf Steiner, PhD, (1861-1925), founder of the Society for Cancer Research, in Arlesheim, Switzerland and introduced in the treatment of human cancer as early as 1921. The anthroposophical preparations are available from different host trees such as oak, apple, pine and others. Harvesting procedure is standardized, and the juices from summer and winter harvests are mixed together. The total extract of mistletoe extracts is considered essential for full effectiveness and concentration of its compounds is ensured through process standardization (http://wissenschaft.misteltherapie.de/- Mistletoe in cancer treatment).

Mistletoe can biosynthesize their own compounds, but it can take some nutrients from the host trees. It has been suggested that pharmacologically active compounds may pass from the host trees to the parasitic plants (Büssing and Schietzel., 1999).

The main ingredients of the *Viscum album* extract are its three ribosome inactivating proteins or lectins (mistletoe lectins, ML) ML-1, ML-2, and ML-3 (Hajtó et al., 2005), the glycoprotein binding with D-galactose and N-acetyl-D-galactosamine, viscotoxins (VT) (Urech et al., 2006), as well as, oligo- and polysaccharides, alkaloids (Khwaja et al., 1980). The flavonoid patterns of *V. album* from various hosts were investigate by Becker and Exner (1980). They identified quercetin and a series of quercetin methyl ethers, which may be assumed to be accumulated on the plant surface. The epicuticular material of the *V. album* contains preferably the flavonol quercetin and its methyl derivatives, occasionally also the flavonol kaempferol and some of its methyl derivatives, and rarely the flavanone naringenin (Haas et al., 2003).

It has been suggested that pharmacologically active compounds may pass from the host trees to the parasitic plants (Büssing and Schietzel., 1999).

In recent years, the research studies were focused on the antioxidant activity of mistletoe (Onay-Ucar et al. 2006; Leu et al., 2006; Yao et al., 2006; Shi et. al., 2006; Vicas et al., 2009b; Choudhary et al., 2010).

Many plant extracts exhibit efficient antioxidant properties due their phytoconstituents, such as phenolics, especially phenolic acids and flavonoids (Aqil *et al.,* 2006; Miliauskas *et al.,* 2004) and carotenoids (Stahl and Sies, 2003).

To evaluate the antioxidant capacities of plant extracts, numerous *in vitro* methods have been developed. DPPH (2,2-diphenyl-1-picrylhydrazyl), ORAC (oxygen radical absorbance capacity), Trolox equivalent antioxidant capacity (TEAC), and ferric-reducing ability (FRAP) are among the more popular methods that have been used (Wu *et al.,* 2004). The advantages and disadvantages of these methods have been fully discussed in several

Antioxidant Activity of European Mistletoe (*Viscum album*) 119

chlorogenic acid, p-OH benzoic acid, caffeic acid, syringic acid, salicilyc acid, p-coumaric acid, ferulic acid, sinapic acid, trans-cinamic acid, naringenin, quercetin, kaempherol and rosmarinic acid. The quantification was made by comparison to calibration curves with pure standards, in the range 0.48 to 500.0 µg/ml. The regression coeficients of calibration curves ranged between 0.9812 and 0.9999. Integration and data analysis were made using

Quantitative data regarding the phenolic compounds composition of mistletoe extracts are

We identified and quantified 17 compounds from mistletoe samples (Tab. 1), including a pentacyclic triterpene (betulinic acid), 12 phenolic acids (gallic acid, protocatechuic acid, gentisic acid, chlorogenic acid, p-OH benzoic acid, caffeic acid, syringic acid, salicilyc acid, p-coumaric acid, ferulic acid, sinapic acid, and trans-cinamic acid) and 4 polyphenols (naringenin, quercetin, kaempherol and rosmarinic acid). The Fig. 3 presents the chemical

These compounds were identified according to their retention time and the spectral characteristics of their peaks compared with standards, as well as by spiking the sample with individual standards. Phenolic compounds are found usually in nature as esters and rarely as glycosides or in free form. Thus, hydrolysis was needed for their identification and quantitative determination. Flavonoids are also present in plants in the form of glycosides. Each flavonoid may occur in a plant in several glycosidic combinations. For this reason, hydrolysis was used to release the aglycones which were further investigated by HPLC.

Quantitative HPLC analysis of *V. album* showed a higher content of bioactive compounds in the leaves compared with stems (Tab. 1). In the case of leaves from *V. album* hosted by *Acer campestre* (VAA), seven phenolic acids and three polyphenols were identified, while in the stem of mistletoe we found only one phenolic acid (trans-cinnamic acid). Caffeic acid was the dominant compound (13.61 µg/g dry matter) in the leaves of mistletoe. Kampherol and rosmarinic acid were presented in both, leaves and stems, while quercetin was identified

The mistletoe hosted by *Fraxinus excelsior* (VAF) contains nine phenolic acids, and two flavonoids. HPLC chromatograms of phenolic acids from leaves and stems of *Viscum album* hosted by *Fraxinus excelsior* are presented in Fig. 4. Concentration of para-coumaric acid in the VAF sample was 1.82 µg/g dry matter, but we have not identified it in other mistletoe extracts. Caffeic acid was found to have the higest values both in leaves (13.98 µg/g dry matter) and stems (15.86 µg/g dry matter). Kaempherol was also present both in leaves (7.30 µg/g dry matter) and stems (3.66 µg/g dry matter), while quercetin was present only

In case of *V. album* collected from *Populus nigra* (VAP), ferrulic acid was a dominant compound in the set of phenolic acids both in leaves (11.52 µg/g dry matter) and in stems (6.14 µg/g dry matter). Salycilic acid was also present in VAP leaves (8.4 µg/g dry matter) and stems (2.3

The HPLC chromatogram of mistletoe hosted by *Mallus domestica* (VAM) showed seven phenolic acids in leaves. Betulinic acid was present only in this mistletoe, both in leaves (1.87 µg/g dry matter) and stems (2.05 µg/g dry matter). Also, like in mistletoe hosted by *P.*

µg/g dry matter), while in the other mistletoe samples it was detected only in leaves.

Origin 7.0 software.

structure of bioactive compounds investigated.

shown in Tab. 1.

only in leaves.

in leaves (6.05 µg/g dry matter).

reviews (Cao and Prior, 1998; Frankel and Meyer, 2000; Prior and Cao, 1999; Sánchez-Moreno, 2002).

The aim of our research studies are focused, the first, on screening of bioactive compounds (phenolic acid and flavonoids) from leaves and stems of *V. album* that are growing on different host trees, and the second we evaluated the antioxidant activity of *V. album'* leaves and stems.

#### **2. HPLC fingerprint of bioactive compounds from** *Viscum album*

To analyze the bioactive compounds, leaves and stems of *V.album* were harvested in July 2009, from five different host trees located in the Borod-Gheghie region, North-West of Romania country. The mean annual rainfall is 500-700 mm/an. The mean annual minimum and maximum temperatures were -22.30C and 350C, respectively (20-yr averages). The mean annual air temperature is 9.2 0C. The area is opened to West, the frequent air mass is oriented to western circulation, transporting oceanic air, cold and humid. Multi-annual average wind speed is 4.1 m/sec. The mistletoe samples have been harvested at the approximate same height, the trees where from it has been harvested having the same soil (brown soil) and climate conditions. The host trees were located in semi-shaded to sunny area (Vicas et al., 2010). The plant materials were labeled according to the host trees, thus: *Acer campestre* (VAA), *Mallus domestica* (VAM), *Fraxinus excelsior* (VAF), *Populus nigra* (VAP) and *Robinia pseudoacacia* (VAR) for easy identification. A voucher specimen of the plants was deposited in the herbarium of the Environmental Protection Faculty from University of Oradea.

The leaves and stems of *V. album* from different host trees were dried rapidly, in an oven at 900C, for 48 hours, in order to prevent enzymatic degradation (Markham, 1982). The dried plant material was stored in a sealed plastic bag for HPLC analysis. After weighing out a portion of the dried material (approximately 1 g), extraction was carried out with ethanol 70% (1:10, w/v). The mixture was stirred for 24 hours in the dark, and then it was centrifuged for 5 minutes, at 3000 rpm. The ethanol fraction of the supernatant was removed using a rotary evaporator. Further, the aqueous extract was subjected to acid hydrolysis (1N HCl) for 2 hours, at 800C. The aglycones were extracted 3 times with ethyl acetate by continuous stirring and then centrifuged at 5000 rpm, for 5 minutes. The solvent was removed by flushing the samples with nitrogen. The residue resulting after evaporation was dissolved in ultrapure water (300 µl), filtered throught 0.45 µm filters (Millex-LG, Millipore), and subjected to HPLC analysis.

A Shimadzu HPLC system equipped with a LC20AT binary pump, a degaser, a SPD-M20A diode array detector (Shimadzu Corp., Kyoto, Japan) and a SUPELCOSIL TM LC-18 column (Sigma-Aldrich Co), 5µm, 25 cm x 4.6 mm was used. Gradient elution was performed with mobile phase A, composed of methanol: acetic acid: double distilled water (10:2:88 v/v/v) and mobile phase B, comprising methanol: acetic acid: double distilled water (90:3:7 v/v/v), at a flow rate of 1.0 ml/min. All solvents were HPLC grade solvents, filtered through a 0.45-*μ*M membrane (Millipore, U.S.A.) and degassed in an ultrasonic bath before use. The chromatograms were monitored at 280 and 360 nm. The following pure standards were used to quantify the bioactive compounds in the leaves and stems of mistletoe: betulinic acid, gallic acid, protocatechuic acid, gentisic acid, Phytochemicals as Nutraceuticals 118 – Global Approaches to Their Role in Nutrition and Health

reviews (Cao and Prior, 1998; Frankel and Meyer, 2000; Prior and Cao, 1999; Sánchez-

The aim of our research studies are focused, the first, on screening of bioactive compounds (phenolic acid and flavonoids) from leaves and stems of *V. album* that are growing on different host trees, and the second we evaluated the antioxidant activity of *V. album'* leaves

To analyze the bioactive compounds, leaves and stems of *V.album* were harvested in July 2009, from five different host trees located in the Borod-Gheghie region, North-West of Romania country. The mean annual rainfall is 500-700 mm/an. The mean annual minimum and maximum temperatures were -22.30C and 350C, respectively (20-yr averages). The mean annual air temperature is 9.2 0C. The area is opened to West, the frequent air mass is oriented to western circulation, transporting oceanic air, cold and humid. Multi-annual average wind speed is 4.1 m/sec. The mistletoe samples have been harvested at the approximate same height, the trees where from it has been harvested having the same soil (brown soil) and climate conditions. The host trees were located in semi-shaded to sunny area (Vicas et al., 2010). The plant materials were labeled according to the host trees, thus: *Acer campestre* (VAA), *Mallus domestica* (VAM), *Fraxinus excelsior* (VAF), *Populus nigra* (VAP) and *Robinia pseudoacacia* (VAR) for easy identification. A voucher specimen of the plants was deposited in the

The leaves and stems of *V. album* from different host trees were dried rapidly, in an oven at 900C, for 48 hours, in order to prevent enzymatic degradation (Markham, 1982). The dried plant material was stored in a sealed plastic bag for HPLC analysis. After weighing out a portion of the dried material (approximately 1 g), extraction was carried out with ethanol 70% (1:10, w/v). The mixture was stirred for 24 hours in the dark, and then it was centrifuged for 5 minutes, at 3000 rpm. The ethanol fraction of the supernatant was removed using a rotary evaporator. Further, the aqueous extract was subjected to acid hydrolysis (1N HCl) for 2 hours, at 800C. The aglycones were extracted 3 times with ethyl acetate by continuous stirring and then centrifuged at 5000 rpm, for 5 minutes. The solvent was removed by flushing the samples with nitrogen. The residue resulting after evaporation was dissolved in ultrapure water (300 µl), filtered throught 0.45 µm filters (Millex-LG,

A Shimadzu HPLC system equipped with a LC20AT binary pump, a degaser, a SPD-M20A diode array detector (Shimadzu Corp., Kyoto, Japan) and a SUPELCOSIL TM LC-18 column (Sigma-Aldrich Co), 5µm, 25 cm x 4.6 mm was used. Gradient elution was performed with mobile phase A, composed of methanol: acetic acid: double distilled water (10:2:88 v/v/v) and mobile phase B, comprising methanol: acetic acid: double distilled water (90:3:7 v/v/v), at a flow rate of 1.0 ml/min. All solvents were HPLC grade solvents, filtered through a 0.45-*μ*M membrane (Millipore, U.S.A.) and degassed in an ultrasonic bath before use. The chromatograms were monitored at 280 and 360 nm. The following pure standards were used to quantify the bioactive compounds in the leaves and stems of mistletoe: betulinic acid, gallic acid, protocatechuic acid, gentisic acid,

**2. HPLC fingerprint of bioactive compounds from** *Viscum album*

herbarium of the Environmental Protection Faculty from University of Oradea.

Millipore), and subjected to HPLC analysis.

Moreno, 2002).

and stems.

chlorogenic acid, p-OH benzoic acid, caffeic acid, syringic acid, salicilyc acid, p-coumaric acid, ferulic acid, sinapic acid, trans-cinamic acid, naringenin, quercetin, kaempherol and rosmarinic acid. The quantification was made by comparison to calibration curves with pure standards, in the range 0.48 to 500.0 µg/ml. The regression coeficients of calibration curves ranged between 0.9812 and 0.9999. Integration and data analysis were made using Origin 7.0 software.

Quantitative data regarding the phenolic compounds composition of mistletoe extracts are shown in Tab. 1.

We identified and quantified 17 compounds from mistletoe samples (Tab. 1), including a pentacyclic triterpene (betulinic acid), 12 phenolic acids (gallic acid, protocatechuic acid, gentisic acid, chlorogenic acid, p-OH benzoic acid, caffeic acid, syringic acid, salicilyc acid, p-coumaric acid, ferulic acid, sinapic acid, and trans-cinamic acid) and 4 polyphenols (naringenin, quercetin, kaempherol and rosmarinic acid). The Fig. 3 presents the chemical structure of bioactive compounds investigated.

These compounds were identified according to their retention time and the spectral characteristics of their peaks compared with standards, as well as by spiking the sample with individual standards. Phenolic compounds are found usually in nature as esters and rarely as glycosides or in free form. Thus, hydrolysis was needed for their identification and quantitative determination. Flavonoids are also present in plants in the form of glycosides. Each flavonoid may occur in a plant in several glycosidic combinations. For this reason, hydrolysis was used to release the aglycones which were further investigated by HPLC.

Quantitative HPLC analysis of *V. album* showed a higher content of bioactive compounds in the leaves compared with stems (Tab. 1). In the case of leaves from *V. album* hosted by *Acer campestre* (VAA), seven phenolic acids and three polyphenols were identified, while in the stem of mistletoe we found only one phenolic acid (trans-cinnamic acid). Caffeic acid was the dominant compound (13.61 µg/g dry matter) in the leaves of mistletoe. Kampherol and rosmarinic acid were presented in both, leaves and stems, while quercetin was identified only in leaves.

The mistletoe hosted by *Fraxinus excelsior* (VAF) contains nine phenolic acids, and two flavonoids. HPLC chromatograms of phenolic acids from leaves and stems of *Viscum album* hosted by *Fraxinus excelsior* are presented in Fig. 4. Concentration of para-coumaric acid in the VAF sample was 1.82 µg/g dry matter, but we have not identified it in other mistletoe extracts. Caffeic acid was found to have the higest values both in leaves (13.98 µg/g dry matter) and stems (15.86 µg/g dry matter). Kaempherol was also present both in leaves (7.30 µg/g dry matter) and stems (3.66 µg/g dry matter), while quercetin was present only in leaves (6.05 µg/g dry matter).

In case of *V. album* collected from *Populus nigra* (VAP), ferrulic acid was a dominant compound in the set of phenolic acids both in leaves (11.52 µg/g dry matter) and in stems (6.14 µg/g dry matter). Salycilic acid was also present in VAP leaves (8.4 µg/g dry matter) and stems (2.3 µg/g dry matter), while in the other mistletoe samples it was detected only in leaves.

The HPLC chromatogram of mistletoe hosted by *Mallus domestica* (VAM) showed seven phenolic acids in leaves. Betulinic acid was present only in this mistletoe, both in leaves (1.87 µg/g dry matter) and stems (2.05 µg/g dry matter).  Bioactive compounds investigated in the leaves and stem of *V.album.*

OH O C OH

Salicylic acid (9)

Sinapic acid (12)

OH OH

Rosmarinic acid (17)

VAF leaves

11 12

9 10

OH O C

identified only in stems.

mAu

HO

OH OH

Cafeic acid (7)

OH O C

*p-*coumaric acid (10)

OH

C O OH CH3

CH3 CH3

CH3 H2 C

Betulinic acid (1)

COOH

OH O C

OH CH3 O OCH 3

H3 CO OH Ferulic acid (11)

> HO HO

Syringic acid (8)

OH O C

> COOH O C O

0 2 4 6 8 10 12 14 16 18 20 22 24

3

5 6

Time (min)

8

7

H 3 CO OH OCH3

CH3 CH3

2011a).

(39.93 µg/g dry matter), which has not been found in the other samples studied.

The main compound in *V. album* hosted by *Robinia pseudoacacia* (VAR) was gallic acid

O

HO

O

Naringenin (14)

OH

We did not detect gentisic acid in any sample, nor naringenin, while quercetin was

3 - protocatechuic acid 5 - chlorogenic acid 6 - para-OH benzoic acid 7 - cafeic acid 8 - syringic acid 9 - salicylic acid 10 - para-coumaric acid 11- ferulic acid 12 - sinapic acid

Fig. 4. HPLC chromatograms, used to fingerprint and evaluate quantitatively phenolic acids from leaves and stems of *V.album* harvested from *Fraxinus excelsior* (VAF) (Vicas et al.,

6.88 µg/g dry matter, respectively) of VAM samples.

Table 1. Quantitative HPLC analysis of bioactive aglicones of phenolics (µg/g dry matter\*) from leaves and stems of *V. album* harvested from different hosts, on July 2009 (Vicas et al.,2011a).

Phytochemicals as Nutraceuticals 120 – Global Approaches to Their Role in Nutrition and Health

±0.03

3.87 ±0.01

4.  Naringenin nd nd nd nd nd nd nd nd nd nd

3.66 ±0.01

±0.01

Total phenolic acids 53.57 1.23 59.56 49.08 30.92 14.23 23.23 16.14 69.18 2.01

Table 1. Quantitative HPLC analysis of bioactive aglicones of phenolics (µg/g dry matter\*) from leaves and stems of *V. album* harvested from different hosts, on July 2009 (Vicas et

nd 3.07

nd 8.4

nd 1.25

2.  Protocatechuic acid

5. Chlorogenic acid 4.70

7. Cafeic Acid 13.61

9. Salicilyc acid 6.70

11. Ferulic acid 7.58

12. Sinapic acid 5.41

15. Quercetin 0.93

16. Kampherol 2.74

17. Rosmarinic acid 1.94

Ratio leaves/stems of phenolic acid

Total phenolic acid (leaves +stem)

Total flavonoids (leaves +stem)

al.,2011a).

10. para-coumaric acid

13. Trans-cinnamic acid

Flavonoids

Polyphenol

6. para-OH benzoic acid

Phenolic acids

VAA *Acer campestre*

1. Betulinic acid nd nd nd 2.35

±0.01

10.16 ±0.1

±0.04

±0.03

±0.001

±0.3

5.41 ±0.04

±0.01

±0.01

±0.002

8. Syringic acid nd nd 1.11

nd nd 5.06

nd 2.74

nd 10.81 ±0.02

nd 13.98 ±0.01

nd 2.70

nd 8.99

nd 12.35 ±0.01

nd 6.05

3.32±0. 001

nd nd 1.82

1.23 ±0.03

*nigra*, ferulic acid was the main compound in leaves and stems (7.81 µg/g dry matter, and 6.88 µg/g dry matter, respectively) of VAM samples.

Fig. 3. Bioactive compounds investigated in the leaves and stem of *V.album.*

The main compound in *V. album* hosted by *Robinia pseudoacacia* (VAR) was gallic acid (39.93 µg/g dry matter), which has not been found in the other samples studied.

We did not detect gentisic acid in any sample, nor naringenin, while quercetin was identified only in stems.

Fig. 4. HPLC chromatograms, used to fingerprint and evaluate quantitatively phenolic acids from leaves and stems of *V.album* harvested from *Fraxinus excelsior* (VAF) (Vicas et al., 2011a).

Antioxidant Activity of European Mistletoe (*Viscum album*) 123

For the determination of antioxidant activity of mistletoe extract through different methods, we used fresh leaves and stems (2 g) that were homogenized with 10 ml distilled water, or with 10 ml 98% ethanol using an Ultra Turax homogenizator, for 1 minute. This mixture was centrifuged (10 000 rpm, at 40C, for 10 minutes) and the supernatants were filtered through a filter paper. The filtrate was used for the antioxidant activity measurements and total

A rapid, simple and inexpensive method to measure antioxidant capacity of plant extracts involves the use of the free radical, 2,2-Diphenyl-1-picrylhydrazyl (DPPH). DPPH is widely used to test the ability of compounds to act as free radical scavengers or hydrogen donors, and to evaluate antioxidant activity of extracts. The reaction involves a colour change from violet to yellow (Fig.5) that can be easily monitored using a spectrophotometer at

The DPPH radical-scavenging activity was determined using the method proposed by Brand-Williams *et al.,* (1995). The reaction was performed in 12 well-plate. A volume of 200 µl sample and 1.4 ml DPPH solution (80 µM) were added to each microplate well. The decrease in the absorbance of the resulting solution was monitored at 515 nm for 30 min. The percentage of scavenging effect of different extracts against DPPH radicals, was

Where, A0 is absorbance of the blank, and AS is absorbance of the samples at 515 nm

Fig. 5. The structure of DPPH radical and its reduction by an antioxidant.

The comparative antioxidant activity of *V.album* leaves hosted by different host trees, evaluated by the DPPH method shown significantly differences (p < 0.001) regarding to DPPH scavenging effect (%) of aqueous and ethanol extracts in leaves and stems (Vicas et

The results showed that DPPH scavenging effect of aqueous extracts from mistletoe leaves varied between 11.49 %, in the case of mistletoe growing on *Robinia pseudoacacia* (VAR), to 2.22 % in the case of VAM (mistletoe growing on *Mallus domestica*). Higher DPPH

DPPH scavenging effect (% ) = [(A0 - A s) x 100 ] / A0 (1)

phenolics content.

515 nm.

al., 2011a).

**3.1 DPPH inhibition by mistletoe extracts**

calculated using the following equation:

Phenolic acids represent the major fraction of bioactive compounds in all *V. album*. A high variability of phenolic acid rations between leaves and stems was observed. While, VAA and VAR had high ratios (43.51:1 and 34.41:1, respectively) the lowest ratios were observed in the case of VAF and VAM (1.21:1 and 1.44:1, respectively).

In our study, the mistletoe hosted by *Fraxinus excelsior* (VAF) proved to be the richest in phenolic acids (108.64 µg/g dry matter), followed by VAR (71.19 µg/g dry matter), VAA (54.80 µg/g dry matter), VAP (45.15 µg/g dry matter) and VAM (39.37 µg/g dry matter).

The total polyphenols from leaves and stems of *V. album* decreased in the following order: VAR VAF VAA VAP VAM.

Luczkiewicz *et al.* (2001), analyzed the phenolic acids present in mistletoe plants hosted by six different hosts. They found that in mistletoe hosted by *Mallus domestica*, the main compound was rosmarinic acid (17.48 mg %), while in mistletoe hosted by *Populus nigra*, the dominant component was chlorogenic acid (12.34 mg %).

Condrat *et al.*, (2009) investigated also nine phanerogam plants (including the European mistletoe) for their flavonoid content and antioxidant activity. Quercetin and kaempferol concentrations were found to be very low in mistletoe extracts (0.20 µmol/g dry matter, and 0.16 µmol/g dry matter, respectively).

Our study revealed that the flavanone naringenin was not present in all the varieties of *Viscum album* investigated. This result is in agreement with the study of Haas *et al.,* (2003) that did not find naringenin in all subspecies of *V. album* analysed, but they found it, rarely, in epicuticular waxes, in *V. cruciatum*. They also found flavonols (quercetin and occasionally kaempherol, along with some of their methyl derivatives) in epicuticular material of *V. album*.

#### **3. Antioxidant activity of** *Viscum album*

Phenolic compounds have attracted the interest of many researchers because they are powerful antioxidants and can protect the human body from oxidative stress. The antioxidant activity of phenolics is mainly due their redox properties.

The present study inquired a variety of *in vitro* tests, based on the capacity to scavenge free radicals. On the basis of the chemical reactions involved, major antioxidant capacity can be divided into two categories: i) hydrogen atom transfer (HAT) and ii) single-electron transfer (SET) reaction – based assay. HAT-based procedures measure the classical ability of an antioxidant to quench free radicals by hydrogen donation (ORAC method):

**X· + AH XH + A·,** where AH = any H donor

SET-based method detects the ability of a potential antioxidant to transfer one electron to reduce a species, including metals, carbonyls, and radicals (TEAC method):

$$\begin{aligned} \mathrm{X^{\cdot}} + \mathrm{AH} &\to \mathrm{X^{\cdot}} + \mathrm{AH^{+}} \\\\ \mathrm{AH^{+}} + \mathrm{H\_{2}O} &\leftrightarrow \mathrm{A^{\cdot}} + \mathrm{H\_{3}O^{+}} \\\\ \mathrm{X^{\cdot}} + \mathrm{H\_{3}O^{+}} &\to \mathrm{XH} + \mathrm{H\_{2}O} \\\\ \mathrm{M(III)} + \mathrm{AH} &\to \mathrm{AH^{+}} + \mathrm{M(II)} \end{aligned}$$

Phytochemicals as Nutraceuticals 122 – Global Approaches to Their Role in Nutrition and Health

Phenolic acids represent the major fraction of bioactive compounds in all *V. album*. A high variability of phenolic acid rations between leaves and stems was observed. While, VAA and VAR had high ratios (43.51:1 and 34.41:1, respectively) the lowest ratios were observed

In our study, the mistletoe hosted by *Fraxinus excelsior* (VAF) proved to be the richest in phenolic acids (108.64 µg/g dry matter), followed by VAR (71.19 µg/g dry matter), VAA (54.80 µg/g dry matter), VAP (45.15 µg/g dry matter) and VAM (39.37 µg/g dry matter). The total polyphenols from leaves and stems of *V. album* decreased in the following order:

Luczkiewicz *et al.* (2001), analyzed the phenolic acids present in mistletoe plants hosted by six different hosts. They found that in mistletoe hosted by *Mallus domestica*, the main compound was rosmarinic acid (17.48 mg %), while in mistletoe hosted by *Populus nigra*, the

Condrat *et al.*, (2009) investigated also nine phanerogam plants (including the European mistletoe) for their flavonoid content and antioxidant activity. Quercetin and kaempferol concentrations were found to be very low in mistletoe extracts (0.20 µmol/g dry matter, and

Our study revealed that the flavanone naringenin was not present in all the varieties of *Viscum album* investigated. This result is in agreement with the study of Haas *et al.,* (2003) that did not find naringenin in all subspecies of *V. album* analysed, but they found it, rarely, in epicuticular waxes, in *V. cruciatum*. They also found flavonols (quercetin and occasionally kaempherol,

Phenolic compounds have attracted the interest of many researchers because they are powerful antioxidants and can protect the human body from oxidative stress. The

The present study inquired a variety of *in vitro* tests, based on the capacity to scavenge free radicals. On the basis of the chemical reactions involved, major antioxidant capacity can be divided into two categories: i) hydrogen atom transfer (HAT) and ii) single-electron transfer (SET) reaction – based assay. HAT-based procedures measure the classical ability of an

**X· + AH XH + A·,** where AH = any H donor SET-based method detects the ability of a potential antioxidant to transfer one electron to

+ AH X- + AH·+

AH·+ + H2O ↔A· + H3O+

X- + H3O+ XH + H2O

M(III) + AH AH+ + M(II)

along with some of their methyl derivatives) in epicuticular material of *V. album*.

antioxidant activity of phenolics is mainly due their redox properties.

antioxidant to quench free radicals by hydrogen donation (ORAC method):

reduce a species, including metals, carbonyls, and radicals (TEAC method):

X·

in the case of VAF and VAM (1.21:1 and 1.44:1, respectively).

dominant component was chlorogenic acid (12.34 mg %).

VAR VAF VAA VAP VAM.

0.16 µmol/g dry matter, respectively).

**3. Antioxidant activity of** *Viscum album*

For the determination of antioxidant activity of mistletoe extract through different methods, we used fresh leaves and stems (2 g) that were homogenized with 10 ml distilled water, or with 10 ml 98% ethanol using an Ultra Turax homogenizator, for 1 minute. This mixture was centrifuged (10 000 rpm, at 40C, for 10 minutes) and the supernatants were filtered through a filter paper. The filtrate was used for the antioxidant activity measurements and total phenolics content.

#### **3.1 DPPH inhibition by mistletoe extracts**

A rapid, simple and inexpensive method to measure antioxidant capacity of plant extracts involves the use of the free radical, 2,2-Diphenyl-1-picrylhydrazyl (DPPH). DPPH is widely used to test the ability of compounds to act as free radical scavengers or hydrogen donors, and to evaluate antioxidant activity of extracts. The reaction involves a colour change from violet to yellow (Fig.5) that can be easily monitored using a spectrophotometer at 515 nm.

The DPPH radical-scavenging activity was determined using the method proposed by Brand-Williams *et al.,* (1995). The reaction was performed in 12 well-plate. A volume of 200 µl sample and 1.4 ml DPPH solution (80 µM) were added to each microplate well. The decrease in the absorbance of the resulting solution was monitored at 515 nm for 30 min. The percentage of scavenging effect of different extracts against DPPH radicals, was calculated using the following equation:

$$\text{DPPH} \text{ scavenging effect} \left(\% \text{ }\right) = \left[ \left( \text{A}\_0 \text{ }\text{-A s} \right) \times 100 \text{ } \right] / \text{ A}\_0 \tag{1}$$

Where, A0 is absorbance of the blank, and AS is absorbance of the samples at 515 nm

Fig. 5. The structure of DPPH radical and its reduction by an antioxidant.

The comparative antioxidant activity of *V.album* leaves hosted by different host trees, evaluated by the DPPH method shown significantly differences (p < 0.001) regarding to DPPH scavenging effect (%) of aqueous and ethanol extracts in leaves and stems (Vicas et al., 2011a).

The results showed that DPPH scavenging effect of aqueous extracts from mistletoe leaves varied between 11.49 %, in the case of mistletoe growing on *Robinia pseudoacacia* (VAR), to 2.22 % in the case of VAM (mistletoe growing on *Mallus domestica*). Higher DPPH

Antioxidant Activity of European Mistletoe (*Viscum album*) 125

The antioxidant activity of the *V. album* extract from *Robinia pseudoacacia* has the highest, especially in the case of DPPH method (11.49 ±0.04 % for VAR leaves aqueous extracts harvesting in July; 76.60± 0.02% for VAR leaves ethanol extracts harvesting December) and

The ORAC method measures antioxidant inhibition of peroxyl radical-induced oxidations, and thus reflects classical radical chain breaking antioxidant activity by hydrogen atom transfer. The ORAC assay was performed essentially as described by Huang *et al.,*(2002). A volume of 150 µl of working solution of sodium fluorescein (4 x 10-3 mM) was added to 25 µl samples, in a 12 well-microplate. The plate was allowed to equilibrate by incubating it for a minimum of 30 minutes in the Synergy™ HT Multi-Detection Microplate Reader (BioTek Instruments, Winooski, VT) at 37°C. Reaction was initiated by the addition of 25 µl of 2,2'-azobis(2-amidino-propane) dihydrochloride (AAPH) solution (153 mM) and the fluorescence was then monitored kinetically with data taken every minute, at 485 nm, 20 nm bandpass excitation filter, and a 528 nm, 20 nm bandpass emission filter. ORAC values were calculated as described by Cao and Prior (1999). The area under the curve (AUC) and the Net AUC of the standards and samples were determined using equations 2 and 3

AUC 0.5 R2 /R1 R3 /R1 R4 /R1 0.5 Rn /R1 (2)

Where R1- fluorescence value at the initiation of reaction and Rn - fluorescence value after

The standard curve was obtained by plotting the Net AUC of different Trolox concentrations against their concentration (6.25 – 100 µM). ORAC values of samples were then calculated automatically using Microsoft Excel to interpolate the sample's Net AUC

The values obtained by ORAC assays varied from 10.73 ± 1.90 mM Trolox equivalents/g fresh matter for the VAP ethanol leaf extract, to 1.52 ± 1.25 mM Trolox equivalents/g fresh matter for the VAM aqueous stem extract. According to the results obtained in the ORAC assay, there was no significant differences between the antioxidant capacity of leaves and stems for all variants of mistletoe investigated, except for the aqueous leaf extracts of VAA *vs* VAM (p < 0.01), and for the aqueous stem extracts of VAA *vs* VAM (p < 0.05). The highest values were recorded in the case of VAA aqueous leaf extract (5.49 mM Trolox equivalents/g fresh matter) and VAP ethanol leaf extract (10.73 mM Trolox equivalents/g

The TEAC is a spectrophotometric method, widely used for the assessment of antioxidant activity of various substances. This method measures the ability of compounds to scavenge the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical cation in relation to Trolox. The blue/green ABTS+ chromophore radical is produced through the reaction

Net AUC AUC – AUC sample blank (3)

this is also in agreement with total phenolic content (Vicas *et al.,* 2011b).

**3.2 ORAC method**

respectively.

30 min.

values against the Trolox standard curve.

fresh matter) (Vicas et al. 2011a).

**3.3 TEAC method**

scavenging effect was observed in the ethanol extracts, with values ranging from 77.19% (VAF) to 50.47 % (VAA).

The DPPH scavenging effect of extracts of mistletoe stems was lower than that of leaf extracts. No antioxidant activity was detected in aqueous extracts of VAF and VAM stems.

In all samples, stem extracts have lower antioxidant activity than the corresponding leaf extracts, also in the case of ethanol extracts.

Similar results were obtained by Önay-Uçar *et al.,(*2006), who investigated the antioxidant activity of methanol extracts of *V.album* grown on different host trees. Their results showed that mistletoe hosted by *Robinia pseudocacia* (VAR) exhibited 73.44% inhibition of DPPH, and mistletoe hosted by *Acer campestre* (VAA) presented 59.52% inhibition of DPPH. The slight differences between our results and theirs can be assigned to the solvent used for extraction and/or to environmental factors.

Sharma and Bhat (2010), showed that the absorbance profiles of DPPH were highest in a buffered methanol solution, followed by methanol and ethanol solutions. Higher absorbance in methanol solutions implies better sensitivity vis-à-vis ethanol solutions of DPPH.

Roman *et al.,* (2009) investigated the efficiency of ultrafiltration process on the antioxidant activity of aqueous extract of *V. album*. The values obtained by the DPPH assay ranged between 66.2% and 88.2% DPPH inhibition for mistletoe concentrated extracts. The correlation coefficient between data of DPPH inhibition and total protein content was 0.94, suggesting that, besides the phenolic compounds of *Viscum* extracts, viscolectins have a great contribution to the radical scavenging activity.

Other research paper (Oluwaseun and Ganiyu, 2008) investigated the antioxidant properties of methanol extracts of *V. album* isolated from cocoa and cashew trees in the South Western part of Nigeria. The scavenging ability of each methanol extract against DPPH followed a dose-dependent pattern (0-10 mg/ml). The free radical scavenging ability of the *V*. *album* extract from cocoa tree performs better than that from cashew tree, a fact that is in agreement with the total phenol content of the two extracts (182 mg /100 g, and 160 mg /100 g, respectively).

Papuc et al., 2010, investigate the free radicals scavenging activity of ethanol extract from mistletoe. The scavenging activity calculated for *V. album* in % inhibition was 7.2%.

When the activities of the same type of mistletoe extracts, collected from the same host tree, but in different seasons, were compared using the DPPH assay, it was found that the antioxidant activity was, in general, higher in spring (Vicaş *et al.,* 2008). The values obtained in May 2008 by the DPPH assay varied from 42.2 % DPPH inhibition for *V. album* growing on *Robinia pseudocacia* (VAR) to 17.4 % DPPH inhibition for *V. album* growing on *Populus nigra* (VAP). In July, the VAR extracts exhibited the highest capacity to scavenge free radicals (46.91%), but the VAA and VAP extracts lost their antioxidant activity. The differences may be explained by the different environmental factors (temperature, water, irradiation, etc.).

The antioxidant activity of the *V. album* extract from *Robinia pseudoacacia* has the highest, especially in the case of DPPH method (11.49 ±0.04 % for VAR leaves aqueous extracts harvesting in July; 76.60± 0.02% for VAR leaves ethanol extracts harvesting December) and this is also in agreement with total phenolic content (Vicas *et al.,* 2011b).

#### **3.2 ORAC method**

Phytochemicals as Nutraceuticals 124 – Global Approaches to Their Role in Nutrition and Health

scavenging effect was observed in the ethanol extracts, with values ranging from 77.19%

The DPPH scavenging effect of extracts of mistletoe stems was lower than that of leaf extracts. No antioxidant activity was detected in aqueous extracts of VAF and VAM

In all samples, stem extracts have lower antioxidant activity than the corresponding leaf

Similar results were obtained by Önay-Uçar *et al.,(*2006), who investigated the antioxidant activity of methanol extracts of *V.album* grown on different host trees. Their results showed that mistletoe hosted by *Robinia pseudocacia* (VAR) exhibited 73.44% inhibition of DPPH, and mistletoe hosted by *Acer campestre* (VAA) presented 59.52% inhibition of DPPH. The slight differences between our results and theirs can be assigned to the solvent used for extraction

Sharma and Bhat (2010), showed that the absorbance profiles of DPPH were highest in a buffered methanol solution, followed by methanol and ethanol solutions. Higher absorbance in methanol solutions implies better sensitivity vis-à-vis ethanol solutions of

Roman *et al.,* (2009) investigated the efficiency of ultrafiltration process on the antioxidant activity of aqueous extract of *V. album*. The values obtained by the DPPH assay ranged between 66.2% and 88.2% DPPH inhibition for mistletoe concentrated extracts. The correlation coefficient between data of DPPH inhibition and total protein content was 0.94, suggesting that, besides the phenolic compounds of *Viscum* extracts, viscolectins have a

Other research paper (Oluwaseun and Ganiyu, 2008) investigated the antioxidant properties of methanol extracts of *V. album* isolated from cocoa and cashew trees in the South Western part of Nigeria. The scavenging ability of each methanol extract against DPPH followed a dose-dependent pattern (0-10 mg/ml). The free radical scavenging ability of the *V*. *album* extract from cocoa tree performs better than that from cashew tree, a fact that is in agreement with the total phenol content of the two extracts (182 mg /100 g, and 160 mg

Papuc et al., 2010, investigate the free radicals scavenging activity of ethanol extract from

When the activities of the same type of mistletoe extracts, collected from the same host tree, but in different seasons, were compared using the DPPH assay, it was found that the antioxidant activity was, in general, higher in spring (Vicaş *et al.,* 2008). The values obtained in May 2008 by the DPPH assay varied from 42.2 % DPPH inhibition for *V. album* growing on *Robinia pseudocacia* (VAR) to 17.4 % DPPH inhibition for *V. album* growing on *Populus nigra* (VAP). In July, the VAR extracts exhibited the highest capacity to scavenge free radicals (46.91%), but the VAA and VAP extracts lost their antioxidant activity. The differences may be explained by the different environmental factors (temperature, water,

mistletoe. The scavenging activity calculated for *V. album* in % inhibition was 7.2%.

(VAF) to 50.47 % (VAA).

extracts, also in the case of ethanol extracts.

great contribution to the radical scavenging activity.

and/or to environmental factors.

stems.

DPPH.

/100 g, respectively).

irradiation, etc.).

The ORAC method measures antioxidant inhibition of peroxyl radical-induced oxidations, and thus reflects classical radical chain breaking antioxidant activity by hydrogen atom transfer. The ORAC assay was performed essentially as described by Huang *et al.,*(2002). A volume of 150 µl of working solution of sodium fluorescein (4 x 10-3 mM) was added to 25 µl samples, in a 12 well-microplate. The plate was allowed to equilibrate by incubating it for a minimum of 30 minutes in the Synergy™ HT Multi-Detection Microplate Reader (BioTek Instruments, Winooski, VT) at 37°C. Reaction was initiated by the addition of 25 µl of 2,2'-azobis(2-amidino-propane) dihydrochloride (AAPH) solution (153 mM) and the fluorescence was then monitored kinetically with data taken every minute, at 485 nm, 20 nm bandpass excitation filter, and a 528 nm, 20 nm bandpass emission filter. ORAC values were calculated as described by Cao and Prior (1999). The area under the curve (AUC) and the Net AUC of the standards and samples were determined using equations 2 and 3 respectively.

$$\text{AUC} = 0.5 + \left( \text{R2/R1} \right) + \left( \text{R3/R1} \right) + \left( \text{R4/R1} \right) + 0.5 \text{(Rn/R1)} \tag{2}$$

$$\text{Net AUC} = \text{AUC}\_{\text{sample}} - \text{AUC}\_{\text{blank}} \tag{3}$$

Where R1- fluorescence value at the initiation of reaction and Rn - fluorescence value after 30 min.

The standard curve was obtained by plotting the Net AUC of different Trolox concentrations against their concentration (6.25 – 100 µM). ORAC values of samples were then calculated automatically using Microsoft Excel to interpolate the sample's Net AUC values against the Trolox standard curve.

The values obtained by ORAC assays varied from 10.73 ± 1.90 mM Trolox equivalents/g fresh matter for the VAP ethanol leaf extract, to 1.52 ± 1.25 mM Trolox equivalents/g fresh matter for the VAM aqueous stem extract. According to the results obtained in the ORAC assay, there was no significant differences between the antioxidant capacity of leaves and stems for all variants of mistletoe investigated, except for the aqueous leaf extracts of VAA *vs* VAM (p < 0.01), and for the aqueous stem extracts of VAA *vs* VAM (p < 0.05). The highest values were recorded in the case of VAA aqueous leaf extract (5.49 mM Trolox equivalents/g fresh matter) and VAP ethanol leaf extract (10.73 mM Trolox equivalents/g fresh matter) (Vicas et al. 2011a).

#### **3.3 TEAC method**

The TEAC is a spectrophotometric method, widely used for the assessment of antioxidant activity of various substances. This method measures the ability of compounds to scavenge the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical cation in relation to Trolox. The blue/green ABTS+ chromophore radical is produced through the reaction

Antioxidant Activity of European Mistletoe (*Viscum album*) 127

(209.51 ± 0.01 mg GAE/ g fresh matter), harvesting in May, while the lowest value was 83.93 ± 0.001 mg GAE/ g fresh matter for VAF, harvesting in July. The mistletoe stem extracts contained lower levels of phenolics, comparing with leaves, in both solvents. In the ethanol extract, the highest phenolic content was found in VAM (58.97 ± 0.009 mg GAE/ g fresh matter), harvesting in December, followed by VAA extract (51.96 ± 0.006 mg GAE/ g fresh matter). These results can be explained by the influence of harvesting time on the chemical

There are many research studies that have established a correlation between the total phenol content of plants and their antioxidant properties (Klçgün and Altner, 2010; Song *et al*.,

Considering the antioxidant potential of European mistletoe components (leaves and stems) due to their content in phenolic derivatives (phenolic acids and flavonoids) and carotenoids, and their specific hydrophilic and lipophilic character, respectively, we measured comparatively the "lipophilic" and hydrophilic" antioxidant capacity, based on the reducing power of such antioxidants against the ferric tripyridyltriazine (Fe(III)-TPTZ) complex (Fig.6). Statistical correlations between their phenolic or carotenoid concentrations and hydrophilic / lipophilic antioxidant activities, in relation to their location (leaves *versus*

Mistletoe extracts for total phenolic content and hydrophilic and lipophilic antioxidant activity were prepared as presented in Fig. 7. Shortly, 10 g leaves or stems were mixed with 25 ml methanol (MeOH), and then the slurries were kept at 40C for 12 hours. After centrifugation for 20 minutes, the supernatant was recovered and stored at – 200 C until the hydrophilic antioxidant activity (HAA) was assayed. The pellet was dissolved in acetone, homogenized and sonicated to extract the lipophilic components submitted to lipophilic antioxidant activity (LAA) analysis. The homogenates were centrifuged for 20 minutes, and

Fig. 6. Reducing power of an antioxidants against the ferric tripyridyltriazine (Fe(III)-TPTZ)

antioxidant

Fe(TPTZ)3(III) + ArOH → Fe (TPTZ)2(II) + ArOH+**.**

The ferric reducing antioxidant power (FRAP) assay was used to determine both hydrophilic and lipophilic antioxidant activities. The assay was determined according to the method of Benzie and Strain (1996) with some modifications. The FRAP assay consists in the ferric tripyridyltriazine (Fe(III)-TPTZ) complex reduction to the ferrous tripyridyltriazine (Fe(II)-TPTZ) by an antioxidant at low pH. The stock solutions included: 300 mM acetate buffer; 250 mg Fe2(SO4)3 · H2O dissolved in 50 ml distillated water; 150 mg TPTZ and 150 µl HCl, dissolved in 50 ml distillated water. The working FRAP solution was freshly prepared

the supernatant was recovered and stored at – 200 C until assayed

composition and antioxidant activity.

2010; Tosun *et al*., 2009; Alali et al., 2007).

**3.5 FRAP method**

stems) are also reported.

complex.

between ABTS and potassium persulfate. In the presence of an antioxidant the ABTS+ radical changes from blue/green to colorless depending on antioxidant capacity of compound, its concentration and the duration of the reaction. ABTS was dissolved in distilled water to a 7 mM concentration. ABTS+ was produced by reacting ABTS stock solution with 2.45 mM potassium persulfate and allowing the mixture to stand, in the dark, at room temperature for 12-16 h before use. ABTS stock solution was diluted with ethanol in order to obtain an absorbance of 0.70 ± 0.02 at 734 nm. After addition of 17 μl of extract to 170 μl of diluted ABTS+, the interaction between the antioxidants and the ABTS·+ was monitored spectrophotometrically at 734 nm (Arnao *et al.* 2001). The results were expressed in mM Trolox equivalent/g fresh matter.

Based upon the conducted research, it has been found that all mistletoe extracts (aqueous or ethanol, leaf or stem) have the ability of scavenging cation-radicals ABTS+. According to the results obtained with TEAC assays, there were significant differences (p < 0.001) between all the extracts investigated (Vicas et al. 2011a). The highest level of scavenging radicals was detected in water extracts, and ethanol extracts had the lowest deactivation level. Aqueous leaf and stem extracts of mistletoe growing on *Acer campestre* (VAA) recorded the highest TEAC values (678.72 ± 0.00 mM equivalent Trolox/g fresh matter, and 577.94 ±0.01 mM equivalent Trolox/g fresh matter, respectively), while for the ethanol extracts the highest level of scavenging cation-radicals ABTS+ was recorded for leaves from VAF (461.09 ±0.11 mM equivalent Trolox/g fresh matter) and for stems from VAP (306.68 ± 0.01 mM equivalent Trolox/g fresh matter). We may suppose that water extracts had the highest antioxidant activity because they contain more bioactive compounds with the ability of scavenging cation-radicals ABTS+, as compared to ethanol extracts (Vicas et al. 2011a).

#### **3.4 Folin-Ciocalteu method**

Total phenolic content was determined by the Folin-Ciocalteu method (Singleton *et al.*, 1999). Mistletoe extract (23 µl) was mixed with 1817 µl distillated water, 115 µl Folin-Ciocalteu reagent (dilution 1:10, v/v) and 345 µl of 15% Na2CO3 solution, and the mixture incubated at room temperature, in the dark, for 2 hours. The absorbance was measured at 765 nm using a spectrophotometer (BioTek Synergy). The calibration curve was linear for the range of concentrations between 0.1-0.5 mg/ml gallic acid. The results were expressed in mg gallic acid equivalents (GAE)/g fresh matter). In aqueous leaf extracts, the highest polyphenolic content was found in VAR (200.51 ± 0.00 mg GAE/g fresh matter, while the lowest value was 176.87 ± 0.003 mg GAE/g fresh matter for VAM. The values obtained for total phenolics in both, aqueous and ethanol extracts, decreased in the order: VAR > VAF > VAP > VAA > VAM. The mistletoe stem extracts contained lower levels of phenolics than the leaf extracts, in both solvents. The lowest level of total phenolics was recorded for VAF and VAA aqueous stem extracts (58% and 54,97% less that leaves, respectively). In the other extracts (VAM or VAR), the differences between leaves and stems were not significant.

In a recent research paper Vicas et al., (2011 b) presented comparatively the total phenolic content from leaves of mistletoe, in three different periods (May, July and December). Generally, the content of total phenolic was higher in aqueous extract comparative with ethanol extract. In aqueous leaves extract, the highest phenolic content was found in VAR (209.51 ± 0.01 mg GAE/ g fresh matter), harvesting in May, while the lowest value was 83.93 ± 0.001 mg GAE/ g fresh matter for VAF, harvesting in July. The mistletoe stem extracts contained lower levels of phenolics, comparing with leaves, in both solvents. In the ethanol extract, the highest phenolic content was found in VAM (58.97 ± 0.009 mg GAE/ g fresh matter), harvesting in December, followed by VAA extract (51.96 ± 0.006 mg GAE/ g fresh matter). These results can be explained by the influence of harvesting time on the chemical composition and antioxidant activity.

There are many research studies that have established a correlation between the total phenol content of plants and their antioxidant properties (Klçgün and Altner, 2010; Song *et al*., 2010; Tosun *et al*., 2009; Alali et al., 2007).

#### **3.5 FRAP method**

Phytochemicals as Nutraceuticals 126 – Global Approaches to Their Role in Nutrition and Health

between ABTS and potassium persulfate. In the presence of an antioxidant the ABTS+ radical changes from blue/green to colorless depending on antioxidant capacity of compound, its concentration and the duration of the reaction. ABTS was dissolved in distilled water to a 7 mM concentration. ABTS+ was produced by reacting ABTS stock solution with 2.45 mM potassium persulfate and allowing the mixture to stand, in the dark, at room temperature for 12-16 h before use. ABTS stock solution was diluted with ethanol in order to obtain an absorbance of 0.70 ± 0.02 at 734 nm. After addition of 17 μl of extract to 170 μl of diluted ABTS+, the interaction between the antioxidants and the ABTS·+ was monitored spectrophotometrically at 734 nm (Arnao *et al.* 2001). The results were expressed

Based upon the conducted research, it has been found that all mistletoe extracts (aqueous or ethanol, leaf or stem) have the ability of scavenging cation-radicals ABTS+. According to the results obtained with TEAC assays, there were significant differences (p < 0.001) between all the extracts investigated (Vicas et al. 2011a). The highest level of scavenging radicals was detected in water extracts, and ethanol extracts had the lowest deactivation level. Aqueous leaf and stem extracts of mistletoe growing on *Acer campestre* (VAA) recorded the highest TEAC values (678.72 ± 0.00 mM equivalent Trolox/g fresh matter, and 577.94 ±0.01 mM equivalent Trolox/g fresh matter, respectively), while for the ethanol extracts the highest level of scavenging cation-radicals ABTS+ was recorded for leaves from VAF (461.09 ±0.11 mM equivalent Trolox/g fresh matter) and for stems from VAP (306.68 ± 0.01 mM equivalent Trolox/g fresh matter). We may suppose that water extracts had the highest antioxidant activity because they contain more bioactive compounds with the ability of scavenging cation-radicals ABTS+, as compared to ethanol extracts (Vicas et al. 2011a).

Total phenolic content was determined by the Folin-Ciocalteu method (Singleton *et al.*, 1999). Mistletoe extract (23 µl) was mixed with 1817 µl distillated water, 115 µl Folin-Ciocalteu reagent (dilution 1:10, v/v) and 345 µl of 15% Na2CO3 solution, and the mixture incubated at room temperature, in the dark, for 2 hours. The absorbance was measured at 765 nm using a spectrophotometer (BioTek Synergy). The calibration curve was linear for the range of concentrations between 0.1-0.5 mg/ml gallic acid. The results were expressed in mg gallic acid equivalents (GAE)/g fresh matter). In aqueous leaf extracts, the highest polyphenolic content was found in VAR (200.51 ± 0.00 mg GAE/g fresh matter, while the lowest value was 176.87 ± 0.003 mg GAE/g fresh matter for VAM. The values obtained for total phenolics in both, aqueous and ethanol extracts, decreased in the order: VAR > VAF > VAP > VAA > VAM. The mistletoe stem extracts contained lower levels of phenolics than the leaf extracts, in both solvents. The lowest level of total phenolics was recorded for VAF and VAA aqueous stem extracts (58% and 54,97% less that leaves, respectively). In the other extracts (VAM or VAR), the differences between leaves and stems were not

In a recent research paper Vicas et al., (2011 b) presented comparatively the total phenolic content from leaves of mistletoe, in three different periods (May, July and December). Generally, the content of total phenolic was higher in aqueous extract comparative with ethanol extract. In aqueous leaves extract, the highest phenolic content was found in VAR

in mM Trolox equivalent/g fresh matter.

**3.4 Folin-Ciocalteu method**

significant.

Considering the antioxidant potential of European mistletoe components (leaves and stems) due to their content in phenolic derivatives (phenolic acids and flavonoids) and carotenoids, and their specific hydrophilic and lipophilic character, respectively, we measured comparatively the "lipophilic" and hydrophilic" antioxidant capacity, based on the reducing power of such antioxidants against the ferric tripyridyltriazine (Fe(III)-TPTZ) complex (Fig.6). Statistical correlations between their phenolic or carotenoid concentrations and hydrophilic / lipophilic antioxidant activities, in relation to their location (leaves *versus*  stems) are also reported.

Mistletoe extracts for total phenolic content and hydrophilic and lipophilic antioxidant activity were prepared as presented in Fig. 7. Shortly, 10 g leaves or stems were mixed with 25 ml methanol (MeOH), and then the slurries were kept at 40C for 12 hours. After centrifugation for 20 minutes, the supernatant was recovered and stored at – 200 C until the hydrophilic antioxidant activity (HAA) was assayed. The pellet was dissolved in acetone, homogenized and sonicated to extract the lipophilic components submitted to lipophilic antioxidant activity (LAA) analysis. The homogenates were centrifuged for 20 minutes, and the supernatant was recovered and stored at – 200 C until assayed

Fig. 6. Reducing power of an antioxidants against the ferric tripyridyltriazine (Fe(III)-TPTZ) complex.

The ferric reducing antioxidant power (FRAP) assay was used to determine both hydrophilic and lipophilic antioxidant activities. The assay was determined according to the method of Benzie and Strain (1996) with some modifications. The FRAP assay consists in the ferric tripyridyltriazine (Fe(III)-TPTZ) complex reduction to the ferrous tripyridyltriazine (Fe(II)-TPTZ) by an antioxidant at low pH. The stock solutions included: 300 mM acetate buffer; 250 mg Fe2(SO4)3 · H2O dissolved in 50 ml distillated water; 150 mg TPTZ and 150 µl HCl, dissolved in 50 ml distillated water. The working FRAP solution was freshly prepared

Antioxidant Activity of European Mistletoe (*Viscum album*) 129

Total phenolics concentration was determined by the Folin-Ciocalteu method, and total carotenoids content from the lipophilic and hydrophilic fraction was determined by the VIS absorption at 470 nm, using a β-carotene (0.001-0.004 mg/ml) standard curve. The total carotenoids content was expressed based on β-carotene equivalents (β-carotene; mg/g fresh

The total phenolics and carotenoids content were measured in methanol and acetone extracts. Methanol extract showed relatively high phenolics content (between 0.65 and 0.40 mg GAE/g fresh weight), which are known as the major natural hydrophilic antioxidants. The phenolics content did not differ significantly between leaves and stems in the methanol extract. But, in the acetone extract the content in the phenolic compounds are 100 time lower comparing to the methanol extract (from 0.015 to 0.002 mg GAE/g FW). Generally, the acetone extracts gave lower values of phenolics and carotenoids than methanol extract. The leaves had, in both solvents, higher concentrations of phenolics and carotenoids, comparing with stems. The stem acetone extract did not contain any carotenoids. Mistletoe leaves originating *Acer campestre* (VAJ), followed by VAM and VAF showed higher concentrations

The HAA values, as determined by FRAP method, were significantly correlated with the values of phenolics content, as determined by Folin-Ciocalteu assay (R2 = 0.9363) in the case of leaves, and R2 = 0.761 in the case of stems, as shown in Fig. 8. The LAA values, as determined by FRAP method, were slightly correlated with the carotenoids content (R2 = 0.6327) (Fig. 8A), and meanwhile HAA were no correlated with carotenoids

The methanol extracts of *V. album* demonstrated to be rich in phenolic compounds, potential antioxidants with ferric reducing ability. Mistletoe leaves originating from *Acer campestre* (VAJ), followed by VAM and VAF showed higher concentrations of phenolics, and also carotenoids, superior to acetone extracts. Meanwhile, VAF and VAM showed higher HAA and LAA activities. These data suggest that the antioxidant capacity slightly differs

Fig. 8. Correlations found between the HAA, as determined by FRAP method, and total phenolics concentration values (GAE units) of mistletoe leaves (A) and stems (B) (Vicas et

Other authors (Onay-Ucar et al., 2006) also reported that antioxidant capacity of *V. album* extract differ depending on the time of harvest and nature of the host trees. Similar results

of phenolics, and also carotenoids especially in methanol.

(R2 = 0.168) (Fig. 8 B) (Vicas et al., 2009b).

depending on the host trees.

al., 2009b).

weight).

by mixing 50 ml acetate buffer, 5 ml Fe2(SO4)3 ·H2O solution and 5 ml TPTZ solution. Mistletoe extracts (100 µl) were allow to react with 500 μl FRAP solution and 2 ml distilled water, for 1 h, in dark. The final colored product (ferrous tripyridyltriazine complex) was quantified by VIS absorption at 595 nm. As positive antioxidant control we used ascorbic acid (AA) and obtained a standard linear curve, between 5 and 100 mg/l vitamin C. The antioxidant activity (HAA and LAA) was expressed in mg/l AA equivalents/ g fresh weight.

We noticed no semnificative differences between the HAA values from leaves and stems hydrophilic fractions, but mistletoe leaves extract originating from *Mallus domestica* (VAM) and *Fraxinus excelsior* (VAF) and all stem extracts have shown the highest antioxidant activity, (0.14 ± 0.12 and 0.13 ± 0.11mg/l vitamin C equivalent / g of fresh leaves).

Meanwhile, LAA is significantly lower (around 100 times) comparing to HAA, in both leaves and stems. No significant differences were noticed between stem and leaves of mistletoe extract. Overall, we observed better antioxidant capacity for VAF and VAM.

A reason for low LAA values (which can be due to low carotenoid concentrations found in acetone extract) can be also the overlapping of carotenoids absorption (450 nm) and the color developed during FRAP method (UV-Vis absorbtion at 595 nm), observed also by other authors who studied vegetable extracts (Ou *et al.,* 2002).

Fig. 7. Flow sheet of extraction of mistletoe extract for the determination of antioxidant activity of hydrophilic and lipophilic fractions (HAA and LAA, respectively). (Vicas et al., 2009b).

Phytochemicals as Nutraceuticals 128 – Global Approaches to Their Role in Nutrition and Health

by mixing 50 ml acetate buffer, 5 ml Fe2(SO4)3 ·H2O solution and 5 ml TPTZ solution. Mistletoe extracts (100 µl) were allow to react with 500 μl FRAP solution and 2 ml distilled water, for 1 h, in dark. The final colored product (ferrous tripyridyltriazine complex) was quantified by VIS absorption at 595 nm. As positive antioxidant control we used ascorbic acid (AA) and obtained a standard linear curve, between 5 and 100 mg/l vitamin C. The antioxidant activity (HAA and LAA) was expressed in mg/l AA equivalents/ g fresh

We noticed no semnificative differences between the HAA values from leaves and stems hydrophilic fractions, but mistletoe leaves extract originating from *Mallus domestica* (VAM) and *Fraxinus excelsior* (VAF) and all stem extracts have shown the highest antioxidant

Meanwhile, LAA is significantly lower (around 100 times) comparing to HAA, in both leaves and stems. No significant differences were noticed between stem and leaves of mistletoe extract. Overall, we observed better antioxidant capacity for VAF and VAM.

A reason for low LAA values (which can be due to low carotenoid concentrations found in acetone extract) can be also the overlapping of carotenoids absorption (450 nm) and the color developed during FRAP method (UV-Vis absorbtion at 595 nm), observed also by

> **MISTLETOE Leaves and stems fresh**

**Supernatant Pellet**

Mixed with 25 ml MeOH

**Supernatant Pellet**

Mixed with acetone

 Sonicate Centrifuged

 Homogenized Kept at 40C, 12 hours Centrifuged

Fig. 7. Flow sheet of extraction of mistletoe extract for the determination of antioxidant activity of hydrophilic and lipophilic fractions (HAA and LAA, respectively). (Vicas et al., 2009b).

**Lipophilic fraction for LAA**

activity, (0.14 ± 0.12 and 0.13 ± 0.11mg/l vitamin C equivalent / g of fresh leaves).

other authors who studied vegetable extracts (Ou *et al.,* 2002).

**Hydrophilic fraction for HAA**

weight.

Total phenolics concentration was determined by the Folin-Ciocalteu method, and total carotenoids content from the lipophilic and hydrophilic fraction was determined by the VIS absorption at 470 nm, using a β-carotene (0.001-0.004 mg/ml) standard curve. The total carotenoids content was expressed based on β-carotene equivalents (β-carotene; mg/g fresh weight).

The total phenolics and carotenoids content were measured in methanol and acetone extracts. Methanol extract showed relatively high phenolics content (between 0.65 and 0.40 mg GAE/g fresh weight), which are known as the major natural hydrophilic antioxidants.

The phenolics content did not differ significantly between leaves and stems in the methanol extract. But, in the acetone extract the content in the phenolic compounds are 100 time lower comparing to the methanol extract (from 0.015 to 0.002 mg GAE/g FW). Generally, the acetone extracts gave lower values of phenolics and carotenoids than methanol extract. The leaves had, in both solvents, higher concentrations of phenolics and carotenoids, comparing with stems. The stem acetone extract did not contain any carotenoids. Mistletoe leaves originating *Acer campestre* (VAJ), followed by VAM and VAF showed higher concentrations of phenolics, and also carotenoids especially in methanol.

The HAA values, as determined by FRAP method, were significantly correlated with the values of phenolics content, as determined by Folin-Ciocalteu assay (R2 = 0.9363) in the case of leaves, and R2 = 0.761 in the case of stems, as shown in Fig. 8. The LAA values, as determined by FRAP method, were slightly correlated with the carotenoids content (R2 = 0.6327) (Fig. 8A), and meanwhile HAA were no correlated with carotenoids (R2 = 0.168) (Fig. 8 B) (Vicas et al., 2009b).

The methanol extracts of *V. album* demonstrated to be rich in phenolic compounds, potential antioxidants with ferric reducing ability. Mistletoe leaves originating from *Acer campestre* (VAJ), followed by VAM and VAF showed higher concentrations of phenolics, and also carotenoids, superior to acetone extracts. Meanwhile, VAF and VAM showed higher HAA and LAA activities. These data suggest that the antioxidant capacity slightly differs depending on the host trees.

Fig. 8. Correlations found between the HAA, as determined by FRAP method, and total phenolics concentration values (GAE units) of mistletoe leaves (A) and stems (B) (Vicas et al., 2009b).

Other authors (Onay-Ucar et al., 2006) also reported that antioxidant capacity of *V. album* extract differ depending on the time of harvest and nature of the host trees. Similar results

Antioxidant Activity of European Mistletoe (*Viscum album*) 131

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were obtained by Oluwaseun and Ganiyu (2007), who evaluated the antioxidant activity of methanol extract of *V. album* leaves from two hosts (cocoa and cashew trees), showing that mistletoe from cocoa tree had higher total phenol content (182 mg/100g) than that from cashew tree (160 mg/100g), the main reason of their antioxidant capacity. Therefore, that the total phenolic content, more than carotenoids content can serve as a useful indicator for the antioxidant activities of mistletoe extracts. Carotenoids are less available also for extraction, being linked to proteins in the photosynthetic apparatus in leaves, a possible.

### **4. Conclusion**

The influence of the host tree may have a key role in the phenolic composition of mistletoe leaves or stems. The mistletoe hosted by *Fraxinus excelsior* (VAF) proved to be the richest in phenolic acids (108.64 µg/g dry matter), followed by VAR (71.19 µg/g dry matter), VAA (54.79 µg/g dry matter), VAP (45.15 µg/g dry matter) and VAM (39.37 µg/g dry matter), as determined by HPLC. The total polyphenols from leaves and stems of *V. album* decreased in the following order: VAR VAF VAA VAP VAM.

In aqueous leaf extracts, the highest polyphenol content was found in VAR (200.51 ± 0.00 mg GAE/ g fresh matter, while the lowest value was 176.87 ± 0.003 mg GAE/ g fresh matter for VAM. The values obtained for total phenols in both, aqueous and ethanol extracts, decreased in the order: VAR > VAF > VAP > VAA > VAM. The mistletoe stem extracts contained lower levels of phenols, as compared to leaves, in both solvents.

The bioactive compounds and the antioxidant activity are present in leaves and also in stems, in all the mistletoe samples examined (aqueous and ethanol). Of the samples examined, the best results were obtained with ethanol extract of VAF, followed by VAR.

As it has been observed by other authors (Cao and Prior, 1998), the values obtained for the antioxidant capacity of an extract depend greatly on the methodology used. The antioxidant potential is reflected by a more complex synergy of active molecules, not only phenols.

The differences in antioxidant activity between leaves and stems of mistletoes harvested from different trees can be attributed to environmental factors such as season, climate and temperature which can significantly affect the accumulation of the antioxidant components in the plant tissue.

#### **5. Acknowledgment**

This work was supported by CNCSIS-UEFISCSU, project number 1120, PN II – IDEI 696/2008, Romania.

#### **6. References**

Alali, F.Q.; Tawaha, K.; El-Elimat, T.; Syouf, M.; El-Fayad, M.; Abulaila, K.; Nielsen, S.J.; Wheaton, W.D.; Falkinham, J.O. 3rd; & Oberlies, N.H. (2007). Antioxidant activity Phytochemicals as Nutraceuticals 130 – Global Approaches to Their Role in Nutrition and Health

were obtained by Oluwaseun and Ganiyu (2007), who evaluated the antioxidant activity of methanol extract of *V. album* leaves from two hosts (cocoa and cashew trees), showing that mistletoe from cocoa tree had higher total phenol content (182 mg/100g) than that from cashew tree (160 mg/100g), the main reason of their antioxidant capacity. Therefore, that the total phenolic content, more than carotenoids content can serve as a useful indicator for the antioxidant activities of mistletoe extracts. Carotenoids are less available also for extraction, being linked to proteins in the photosynthetic apparatus in leaves, a

The influence of the host tree may have a key role in the phenolic composition of mistletoe leaves or stems. The mistletoe hosted by *Fraxinus excelsior* (VAF) proved to be the richest in phenolic acids (108.64 µg/g dry matter), followed by VAR (71.19 µg/g dry matter), VAA (54.79 µg/g dry matter), VAP (45.15 µg/g dry matter) and VAM (39.37 µg/g dry matter), as determined by HPLC. The total polyphenols from leaves and stems of *V. album* decreased in

In aqueous leaf extracts, the highest polyphenol content was found in VAR (200.51 ± 0.00 mg GAE/ g fresh matter, while the lowest value was 176.87 ± 0.003 mg GAE/ g fresh matter for VAM. The values obtained for total phenols in both, aqueous and ethanol extracts, decreased in the order: VAR > VAF > VAP > VAA > VAM. The mistletoe stem extracts

The bioactive compounds and the antioxidant activity are present in leaves and also in stems, in all the mistletoe samples examined (aqueous and ethanol). Of the samples examined, the best results were obtained with ethanol extract of VAF, followed by VAR.

As it has been observed by other authors (Cao and Prior, 1998), the values obtained for the antioxidant capacity of an extract depend greatly on the methodology used. The antioxidant potential is reflected by a more complex synergy of active molecules, not only

The differences in antioxidant activity between leaves and stems of mistletoes harvested from different trees can be attributed to environmental factors such as season, climate and temperature which can significantly affect the accumulation of the antioxidant components

This work was supported by CNCSIS-UEFISCSU, project number 1120, PN II – IDEI

Alali, F.Q.; Tawaha, K.; El-Elimat, T.; Syouf, M.; El-Fayad, M.; Abulaila, K.; Nielsen, S.J.;

Wheaton, W.D.; Falkinham, J.O. 3rd; & Oberlies, N.H. (2007). Antioxidant activity

contained lower levels of phenols, as compared to leaves, in both solvents.

the following order: VAR VAF VAA VAP VAM.

possible.

phenols.

in the plant tissue.

696/2008, Romania.

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**5. Acknowledgment**

**4. Conclusion**

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**8**

**Antioxidant and Anti-Inflammatory Activities**

Oxidative stress plays a critical role in the pathogenesis of inflammation (Winrow et al., 1993), which is a physiological response that protects the body from stimuli including infections and tissue injury. The magnitude of the inflammatory response is crucial, and insufficient responses result in immunodeficiency, which can lead to infection and cancer. Excessive responses cause morbidity and mortality from diseases such as rheumatoid arthritis, Crohn's disease, atherosclerosis, diabetes, Alzheimer's disease, multiple sclerosis, and cerebral and myocardial ischemia (Tracey, 2002). Inflammation is associated with a wide range of inflammatory mediators that initiate inflammatory responses, recruit and activate other cells to the site of inflammation, and subsequently resolve the inflammation (Gallin & Snyderman, 1999). The expression of pro-inflammatory mediators such as cytokines, chemokines, adhesion molecules, iNOS, and COX-2 involves nuclear factor-κB (NF-κB) (Baeuerle & Baltimore, 1996; Hayden & Ghosh, 2004). Mitogen-activated protein kinases (MAPKs) pathways are also reportedly stimulated by inflammatory mediators

The genus *Sasa* (Poaceae) is composed of perennial plants commonly known as bamboo grasses, and various *Sasa* species are widely distributed in Asian countries including China, Japan, Korea, and Russia (Okabe et al., 1975). *Sasa* leaves have been used in traditional medicine for their anti-inflammatory, antipyretic, and diuretic properties (Bae, 2000). Bamboo leaves have also been used in clinical settings to treat hypertension, cardiovascular

Many recent studies have described the beneficial health effects of *Sasa* species leaves, which have been used as alternative medicines. *S. albomarginata* extract reportedly has anticancer properties (Shibata et al., 1979). Both lignin and polysaccharide preparation from *Sasa* species reportedly have antitumor properties (Suzuki et al., 1968; Yamafuji & Murakami, 1968). Two polysaccharide preparations (GK1 and GK2) from *S. kurilensis* was found to negatively affect the growth of Sarcoma-180 implanted in mice (Raidaru et al., 1997). Also, Sasa Health, an alkaline extract derived from *S. senanensis* leaves containing polysaccharides, chlorophyllin, lignin, and flavonoids, reportedly has a protective effect on

**1. Introduction**

(Guha & Mackman, 2001).

disease, and cancer (Shibata et al., 1975).

**of** *Sasa quelpaertensis* **Leaf Extracts**

Se-Jae Kim, Joon-Ho Hwang, Hye-Sun Shin,

 *Jeju National University, Jeju,*

*Republic of Korea*

Mi-Gyeong Jang, Hee-Chul Ko and Seong-Il Kang *Department of Biology and Sasa Industry Development Agency,*

Yao, H.; Liao, Z-X.; Wu, Q.; Lei, G-Q.; Liu, Z-J.; Chen, D-F.; Chen, J-K.; Zhou, T-S. (2006). Antioxidative flavanone glycosides from the branches and leaves of *Viscum coloratum*. *Chemical and Pharmaceutical Bulletin,* Vol. 54, pp. 133-135, ISSN 0009- 2363

### **Antioxidant and Anti-Inflammatory Activities of** *Sasa quelpaertensis* **Leaf Extracts**

Se-Jae Kim, Joon-Ho Hwang, Hye-Sun Shin, Mi-Gyeong Jang, Hee-Chul Ko and Seong-Il Kang *Department of Biology and Sasa Industry Development Agency, Jeju National University, Jeju, Republic of Korea*

#### **1. Introduction**

Phytochemicals as Nutraceuticals 134 – Global Approaches to Their Role in Nutrition and Health

Yao, H.; Liao, Z-X.; Wu, Q.; Lei, G-Q.; Liu, Z-J.; Chen, D-F.; Chen, J-K.; Zhou, T-S. (2006).

2363

Antioxidative flavanone glycosides from the branches and leaves of *Viscum coloratum*. *Chemical and Pharmaceutical Bulletin,* Vol. 54, pp. 133-135, ISSN 0009-

> Oxidative stress plays a critical role in the pathogenesis of inflammation (Winrow et al., 1993), which is a physiological response that protects the body from stimuli including infections and tissue injury. The magnitude of the inflammatory response is crucial, and insufficient responses result in immunodeficiency, which can lead to infection and cancer. Excessive responses cause morbidity and mortality from diseases such as rheumatoid arthritis, Crohn's disease, atherosclerosis, diabetes, Alzheimer's disease, multiple sclerosis, and cerebral and myocardial ischemia (Tracey, 2002). Inflammation is associated with a wide range of inflammatory mediators that initiate inflammatory responses, recruit and activate other cells to the site of inflammation, and subsequently resolve the inflammation (Gallin & Snyderman, 1999). The expression of pro-inflammatory mediators such as cytokines, chemokines, adhesion molecules, iNOS, and COX-2 involves nuclear factor-κB (NF-κB) (Baeuerle & Baltimore, 1996; Hayden & Ghosh, 2004). Mitogen-activated protein kinases (MAPKs) pathways are also reportedly stimulated by inflammatory mediators (Guha & Mackman, 2001).

> The genus *Sasa* (Poaceae) is composed of perennial plants commonly known as bamboo grasses, and various *Sasa* species are widely distributed in Asian countries including China, Japan, Korea, and Russia (Okabe et al., 1975). *Sasa* leaves have been used in traditional medicine for their anti-inflammatory, antipyretic, and diuretic properties (Bae, 2000). Bamboo leaves have also been used in clinical settings to treat hypertension, cardiovascular disease, and cancer (Shibata et al., 1975).

> Many recent studies have described the beneficial health effects of *Sasa* species leaves, which have been used as alternative medicines. *S. albomarginata* extract reportedly has anticancer properties (Shibata et al., 1979). Both lignin and polysaccharide preparation from *Sasa* species reportedly have antitumor properties (Suzuki et al., 1968; Yamafuji & Murakami, 1968). Two polysaccharide preparations (GK1 and GK2) from *S. kurilensis* was found to negatively affect the growth of Sarcoma-180 implanted in mice (Raidaru et al., 1997). Also, Sasa Health, an alkaline extract derived from *S. senanensis* leaves containing polysaccharides, chlorophyllin, lignin, and flavonoids, reportedly has a protective effect on

Antioxidant and Anti-Inflammatory Activities of *Sasa quelpaertensis* Leaf Extracts 137

(MeOH) at room temperature for 48 h. This procedure was repeated twice. The combined extract was concentrated on a rotary evaporator under reduced pressure and freeze-dried to a powder. The dried extract was dissolved in water and then fractionated using the organic solvents *n*-hexane (hexane), ethyl acetate (EtOAc), and *n*-butanol (BuOH) at room temperature. Each fraction was concentrated on a rotary evaporator under reduced pressure

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was examined according to the method reported by Tateyama et al. (1997) with slight modifications. Briefly, the extracts were mixed with methanol and then added to 0.4 mM DPPH in methanol. After 20 min of incubation in the dark at room temperature, the reduction in the DPPH free radical was measured by absorbance, which was read using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA) at 517 nm. Trolox and BHA, a stable antioxidant, were used as a reference control and pure methanol was used as a control sample. Three replicates were made for each test sample. The radical scavenging activity of samples, expressed as percent inhibition, was calculated according to the follow formula: % inhibition = (*A*0/*A*X)/*A*0 100, where *A*0 and *A*X are the absorbance values of the blank sample and the tested samples, respectively. The results were indicated as IC50, which is the 50%

Nitric oxide (NO) scavenging activity was measured using the method described by Feelisch and Stamler (1996) with slight modifications. Sodium nitroprusside (SNP) in an aqueous solution at physiological pH spontaneously generates nitric oxide, which can be measured using the Griess reagent system. The reaction solution containing 1 mM SNP in phosphatebuffered saline (PBS) (pH 7.4) was mixed with the extract, followed by incubation at room temperature for 3 h. Then, the reaction solution was mixed with an equal volume of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine in 5% phosphoric acid). The absorbance at 540 nm was recorded using a microplate reader. The percentage of NO scavenging was measured through comparison with the absorbance values of the blank

Superoxide (O2–) was generated using the enzymatic method. Briefly, the extracts were mixed with an equal volume of reaction reagent [2 mM Na2 EDTA in phosphate buffer (50 mM KH2PO4/KOH, pH 7.4), 0.05 mM nitroblue tetrazolium chloride (NBT) in a buffer, and 1 mM hypoxanthine in 50 mM KOH]. The reaction was started by adding xanthine oxidase (XOD) in a buffer to the mixture (final XOD concentration was 0.05 U/ml). After 1 h of incubation at room temperature, scavenging activity was measured by absorbance, which was read using a microplate reader at 560 nm. Trolox and BHA, a stable antioxidant, were used as a reference control; SOD and allopurinol were used as a positive control. The NBT reduction (%) was calculated according to the following formula: % inhibition =

and freeze-dried to a powder.

**2.3.2 NO scavenging activity**

**2.3.3 Superoxide radical scavenging assay**

sample.

**2.3 Measurement of antioxidant activities 2.3.1 DPPH radical scavenging assay**

inhibitory concentration of DPPH radical scavenging activity.

spontaneous mammary tumorigenesis (Tsunoda et al., 1998) and Her2/NeuN mammary tumorigenesis (Ren et al., 2004). Researchers have also recently reported that *S. senanensis* leaf extracts have antioxidant and immunostimulation-mediated antitumor properties (Kurokawa et al., 2006; Seki et al., 2008). Hagasewa et al. (2008) reported antioxidant *C*glycosyl flavones in the leaves of *S. kurilensis* var. *gigantea*. Extract from *S. borealis* leaves reportedly improves chronic high glucose-induced endothelial apoptosis (Choi et al., 2008), as well as insulin resistance by modulating inflammatory cytokine secretion in high fat dietinduced obese C57/BL6J mice (Yang et al., 2010). Park et al. (2007) reported four antioxidant flavone glycosides (tricine-7-*O*-β-D-glucopyranoside, isoorientin, apigenin 6-C-β-Dxylopyranosyl-8-C-β-D-glucopyranoside, isoorientin 2-*O*-α-L-rhamnoside) from *S. borealis*. Two phenolic compounds, (–)-syringaresinol and tricin, isolated from *S. borealis*, exhibited P-glycoprotein inhibitory properties in adrimiamycin-resistant human breast cancer, MCF-7/ARD (Jeong et al., 2007).

*S. quelpaertensis* Nakai is another bamboo grass. It is a native Korean plant that grows only on Mt. Halla on Jeju Island, South Korea. This small bamboo grass has recently been the focus of much attention due to its potential biomass as well as its role as an invasive plant that inhibits the growth of other plants in the habitat on Jeju Island. Young leaves of *S. quelpaertensis* are used for a popular bamboo tea, but their beneficial health effects and the bioactive compounds contained in the plant have not yet been identified. Thus, systematic research about using its leaves as an industrial bio-resource is increasingly required. As a first step to evaluating the potential of *S. quelpaertensis* leaves as nutraceuticals, in this study, we investigated the anti-oxidative and anti-inflammatory activities of *S.quelpaertensis* leaf extract.

#### **2. Materials and methods**

#### **2.1 Reagents**

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin– streptomycin (PS) were obtained from Gibco-BRL (Grand Island, NY, USA). Antibody against inducible NOS (iNOS) was purchased from Calbiochem (San Diego, CA, USA), and antibody against cyclooxygenase-2 (COX-2) was obtained from Becton Dickinson (Mountain View, CA, USA). Anti-phospho-extracellular signal-regulated kinase (ERK1/2) was acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against ERK1/2, JNK1/2, phospho-JNK1/2, p38, and phospho-p38 were obtained from Cell Signaling Technology (Beverly, MA, USA). The lactate dehydrogenase (LDH) Cytotoxicity Detection Kit was purchased from Takara Shuzo Co. (Otsu, Shiga, Japan). Protein assay reagent was purchased from Bio-Rad Laboratories Inc. (Hercules, CA, USA). Trizol reagent was purchased from Molecular Research Center Inc. (Cincinnati, OH, USA), lipopolysaccharide (LPS) (*Escherichia coli* 026:B6) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). All other reagents were acquired from Sigma.

#### **2.2 Preparation of extracts**

*S. quelpaertensis* leaves were collected in October 2010, from Mt. Halla on Jeju Island, South Korea. A dried powder of the *S. quelpaertensis* leaves was extracted using 80% methanol (MeOH) at room temperature for 48 h. This procedure was repeated twice. The combined extract was concentrated on a rotary evaporator under reduced pressure and freeze-dried to a powder. The dried extract was dissolved in water and then fractionated using the organic solvents *n*-hexane (hexane), ethyl acetate (EtOAc), and *n*-butanol (BuOH) at room temperature. Each fraction was concentrated on a rotary evaporator under reduced pressure and freeze-dried to a powder.

#### **2.3 Measurement of antioxidant activities**

#### **2.3.1 DPPH radical scavenging assay**

Phytochemicals as Nutraceuticals 136 – Global Approaches to Their Role in Nutrition and Health

spontaneous mammary tumorigenesis (Tsunoda et al., 1998) and Her2/NeuN mammary tumorigenesis (Ren et al., 2004). Researchers have also recently reported that *S. senanensis* leaf extracts have antioxidant and immunostimulation-mediated antitumor properties (Kurokawa et al., 2006; Seki et al., 2008). Hagasewa et al. (2008) reported antioxidant *C*glycosyl flavones in the leaves of *S. kurilensis* var. *gigantea*. Extract from *S. borealis* leaves reportedly improves chronic high glucose-induced endothelial apoptosis (Choi et al., 2008), as well as insulin resistance by modulating inflammatory cytokine secretion in high fat dietinduced obese C57/BL6J mice (Yang et al., 2010). Park et al. (2007) reported four antioxidant flavone glycosides (tricine-7-*O*-β-D-glucopyranoside, isoorientin, apigenin 6-C-β-Dxylopyranosyl-8-C-β-D-glucopyranoside, isoorientin 2-*O*-α-L-rhamnoside) from *S. borealis*. Two phenolic compounds, (–)-syringaresinol and tricin, isolated from *S. borealis*, exhibited P-glycoprotein inhibitory properties in adrimiamycin-resistant human breast cancer, MCF-

*S. quelpaertensis* Nakai is another bamboo grass. It is a native Korean plant that grows only on Mt. Halla on Jeju Island, South Korea. This small bamboo grass has recently been the focus of much attention due to its potential biomass as well as its role as an invasive plant that inhibits the growth of other plants in the habitat on Jeju Island. Young leaves of *S. quelpaertensis* are used for a popular bamboo tea, but their beneficial health effects and the bioactive compounds contained in the plant have not yet been identified. Thus, systematic research about using its leaves as an industrial bio-resource is increasingly required. As a first step to evaluating the potential of *S. quelpaertensis* leaves as nutraceuticals, in this study, we investigated the anti-oxidative and anti-inflammatory activities of *S.quelpaertensis* leaf

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin– streptomycin (PS) were obtained from Gibco-BRL (Grand Island, NY, USA). Antibody against inducible NOS (iNOS) was purchased from Calbiochem (San Diego, CA, USA), and antibody against cyclooxygenase-2 (COX-2) was obtained from Becton Dickinson (Mountain View, CA, USA). Anti-phospho-extracellular signal-regulated kinase (ERK1/2) was acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against ERK1/2, JNK1/2, phospho-JNK1/2, p38, and phospho-p38 were obtained from Cell Signaling Technology (Beverly, MA, USA). The lactate dehydrogenase (LDH) Cytotoxicity Detection Kit was purchased from Takara Shuzo Co. (Otsu, Shiga, Japan). Protein assay reagent was purchased from Bio-Rad Laboratories Inc. (Hercules, CA, USA). Trizol reagent was purchased from Molecular Research Center Inc. (Cincinnati, OH, USA), lipopolysaccharide (LPS) (*Escherichia coli* 026:B6) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). All

*S. quelpaertensis* leaves were collected in October 2010, from Mt. Halla on Jeju Island, South Korea. A dried powder of the *S. quelpaertensis* leaves was extracted using 80% methanol

7/ARD (Jeong et al., 2007).

**2. Materials and methods**

other reagents were acquired from Sigma.

**2.2 Preparation of extracts**

extract.

**2.1 Reagents**

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was examined according to the method reported by Tateyama et al. (1997) with slight modifications. Briefly, the extracts were mixed with methanol and then added to 0.4 mM DPPH in methanol. After 20 min of incubation in the dark at room temperature, the reduction in the DPPH free radical was measured by absorbance, which was read using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA) at 517 nm. Trolox and BHA, a stable antioxidant, were used as a reference control and pure methanol was used as a control sample. Three replicates were made for each test sample. The radical scavenging activity of samples, expressed as percent inhibition, was calculated according to the follow formula: % inhibition = (*A*0/*A*X)/*A*0 100, where *A*0 and *A*X are the absorbance values of the blank sample and the tested samples, respectively. The results were indicated as IC50, which is the 50% inhibitory concentration of DPPH radical scavenging activity.

#### **2.3.2 NO scavenging activity**

Nitric oxide (NO) scavenging activity was measured using the method described by Feelisch and Stamler (1996) with slight modifications. Sodium nitroprusside (SNP) in an aqueous solution at physiological pH spontaneously generates nitric oxide, which can be measured using the Griess reagent system. The reaction solution containing 1 mM SNP in phosphatebuffered saline (PBS) (pH 7.4) was mixed with the extract, followed by incubation at room temperature for 3 h. Then, the reaction solution was mixed with an equal volume of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine in 5% phosphoric acid). The absorbance at 540 nm was recorded using a microplate reader. The percentage of NO scavenging was measured through comparison with the absorbance values of the blank sample.

#### **2.3.3 Superoxide radical scavenging assay**

Superoxide (O2–) was generated using the enzymatic method. Briefly, the extracts were mixed with an equal volume of reaction reagent [2 mM Na2 EDTA in phosphate buffer (50 mM KH2PO4/KOH, pH 7.4), 0.05 mM nitroblue tetrazolium chloride (NBT) in a buffer, and 1 mM hypoxanthine in 50 mM KOH]. The reaction was started by adding xanthine oxidase (XOD) in a buffer to the mixture (final XOD concentration was 0.05 U/ml). After 1 h of incubation at room temperature, scavenging activity was measured by absorbance, which was read using a microplate reader at 560 nm. Trolox and BHA, a stable antioxidant, were used as a reference control; SOD and allopurinol were used as a positive control. The NBT reduction (%) was calculated according to the following formula: % inhibition =

Antioxidant and Anti-Inflammatory Activities of *Sasa quelpaertensis* Leaf Extracts 139

quantified by enzyme-linked immunosorbent assay (ELISA) according to the

The activity of the iNOS enzyme in the cell lysate was measured as the L-arginine- and NADPH-dependent generation of nitrite. Briefly, 200 µg cell lysate was incubated for 180 min at room temperature in 100 µl of a reaction buffer containing 20 mM sodium phosphate buffer, 2 mM NADPH, 2 mM L-arginine, and 10 µm FAD at pH 6.7. The reaction was stopped by adding 10 U/ml LDH and 10 mM pyruvate. Next, the reaction mixture was incubated with an equal volume of Griess reagent. The absorbance at 540 nm was recorded using a microplate reader. Nitrite concentration was determined by extrapolation from a

Cells were washed twice with ice-cold PBS and collected. The cells were then treated with a lysis buffer [1× RIPA (Upstate Cell Signaling Solutions, Lake Placid, NY, USA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na3VO4, 1 mM NaF, 1 μg/ml aprotinin, 1 μg/ml pepstatin, and 1 μg/ml leupeptin] and incubated on ice for 1 h. Cell debris was removed by centrifugation and then protein concentration was determined using a Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Cell lysates were subjected to 7.5% or 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with a solution of 0.1% Tween 20/Tris-buffed saline containing 5% nonfat milk powder for 1 h at room temperature. After incubation overnight at 4°C with the indicated primary antibody, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Immunodetection was carried out using an ECL Western blotting detection reagent. The signal intensity of relative bands was determined using

**2.9 RNA preparation and reverse transcription-polymerase chain reaction (RT-PCR)**  Total cellular RNA was isolated using a Trizol reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's instructions. RNA concentration and purity were determined by measuring absorbance at 260 and 280 nm. A cDNA synthesis was performed using the ImProm-II™ Reverse Transcription System (Promega, Madison, WI, USA) with an oligo dT-15 primer, as recommended by the supplier. PCR analyses were then performed on aliquots of cDNA preparations using a thermal cycler. The reactions were carried out in a 25 l volume containing (final concentration) 1 unit of Taq DNA polymerase, 0.2 mM dNTP, 10 reaction buffer, and 100 pmol primers (Table 1). The cycle number was optimized to ensure product accumulation in the exponential range. -Actin was used as an internal control to normalize the RNA content of each sample. Amplification was initiated at 94°C for 5 min, followed by 18–27 cycles of denaturation at 94°C for 45 s, annealing at the appropriate primer-pair annealing temperature for 45 s, and extension at 72°C for 1 min, followed by a final extension of 10 min at 72°C. After amplification, portions of the PCR reactions were electrophoresed on 2% agarose gel and visualized using ethidium

image acquisition and analysis software (LabWorks, Cambridge, UK).

manufacturer's instructions.

sodium nitrite standard curve.

**2.8 Western blot analysis**

**2.7 Measurement of iNOS enzyme activity**

(*A*0/*A*X)/*A*0 100, where *A*0 and *A*X are the absorbance values of the blank sample and the tested samples, respectively.

#### **2.3.4 Determination of XOD-inhibitory activity**

XOD-inhibitory activity was measured by detecting uric acid formation according to the method described by Puig et al. (1989) with slight modifications. First, the extracts and reaction reagent [2 mM Na2 EDTA in a phosphate buffer (50 mM KH2PO4/KOH, pH 7.4) and 1 mM hypoxanthine in 50 mM KOH] were mixed in a 96-well microplate. The reaction was started by adding XOD in a buffer to the mixture (final XOD concentration was 0.05 U/ml). After 1 h of incubation at room temperature, uric acid production was measured by absorbance, which was read using a microplate reader at 295 nm. Trolox and BHA, a stable antioxidant, were used as a reference control. SOD and allopurinol were used as a positive control. Uric acid production was calculated according to the following formula: % inhibition = (*A*0/*A*X)/*A*0 100, where *A*0 and *A*X are the absorbance values of the blank sample and the tested samples, respectively.

#### **2.4 Cell culture**

The RAW 264.7 murine macrophage cell line was obtained from the Korea Cell Line Bank (Seoul, Korea). Cells were cultured in 1% PS/DMEM containing 10% heat-inactivated FBS at 37°C in a 5% CO2 incubator.

#### **2.5 Cytotoxicity assay - MTT and LDH release assays**

Cell viability and cytotoxicity were determined using a 3-*(4,5-dimethylthiazol-2-yl)*-2,5 diphenyltetrazolium bromide (MTT) cell viability assay and lactic dehydrogenase (LDH) release assay. Cells were seeded at a density of 5 × 104 cells/well into a 96-well flat-bottom cell culture plate in the presence or absence of the extracts. Mitochondrial enzyme activity, which is an indirect measure of the number of viable respiring cells, was determined using an MTT reagent after 40 h of treatment with methanol extract or its fractions. Absorbance was read using a microplate reader (Bio-Tek Instruments) at 595 nm. The effect of extracts on cell viability was evaluated as the relative absorbance compared to that of control cultures. LDH leakage is known to be a marker of damage to the cell membrane, and the LDH level was detected using the culture supernatants according to the LDH cytotoxicity detection kit instructions (Takara Shuzo Co.). Cytotoxicity was expressed as the percentage of released LDH (LDH released into the medium/maximal LDH release 100). Maximal LDH release was measured after lysis of the cells with 0.5% Triton X-100.

#### **2.6 Measurement of NO and PGE2 production**

The amount of nitrite was determined using a colorimetric assay (Green et al., 1982). Briefly, 100 μl of cell culture medium was mixed with an equal volume of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine in 5% phosphoric acid) and incubated at room temperature for 10 min. The absorbance at 540 nm was recorded using a microplate reader. Nitrite concentration was determined using extrapolation from a sodium nitrite standard curve. Concentration of prostaglandin E2 (PGE2) in the culture medium was quantified by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions.

#### **2.7 Measurement of iNOS enzyme activity**

The activity of the iNOS enzyme in the cell lysate was measured as the L-arginine- and NADPH-dependent generation of nitrite. Briefly, 200 µg cell lysate was incubated for 180 min at room temperature in 100 µl of a reaction buffer containing 20 mM sodium phosphate buffer, 2 mM NADPH, 2 mM L-arginine, and 10 µm FAD at pH 6.7. The reaction was stopped by adding 10 U/ml LDH and 10 mM pyruvate. Next, the reaction mixture was incubated with an equal volume of Griess reagent. The absorbance at 540 nm was recorded using a microplate reader. Nitrite concentration was determined by extrapolation from a sodium nitrite standard curve.

#### **2.8 Western blot analysis**

Phytochemicals as Nutraceuticals 138 – Global Approaches to Their Role in Nutrition and Health

(*A*0/*A*X)/*A*0 100, where *A*0 and *A*X are the absorbance values of the blank sample and the

XOD-inhibitory activity was measured by detecting uric acid formation according to the method described by Puig et al. (1989) with slight modifications. First, the extracts and reaction reagent [2 mM Na2 EDTA in a phosphate buffer (50 mM KH2PO4/KOH, pH 7.4) and 1 mM hypoxanthine in 50 mM KOH] were mixed in a 96-well microplate. The reaction was started by adding XOD in a buffer to the mixture (final XOD concentration was 0.05 U/ml). After 1 h of incubation at room temperature, uric acid production was measured by absorbance, which was read using a microplate reader at 295 nm. Trolox and BHA, a stable antioxidant, were used as a reference control. SOD and allopurinol were used as a positive control. Uric acid production was calculated according to the following formula: % inhibition = (*A*0/*A*X)/*A*0 100, where *A*0 and *A*X are the absorbance values of the blank

The RAW 264.7 murine macrophage cell line was obtained from the Korea Cell Line Bank (Seoul, Korea). Cells were cultured in 1% PS/DMEM containing 10% heat-inactivated FBS at

Cell viability and cytotoxicity were determined using a 3-*(4,5-dimethylthiazol-2-yl)*-2,5 diphenyltetrazolium bromide (MTT) cell viability assay and lactic dehydrogenase (LDH) release assay. Cells were seeded at a density of 5 × 104 cells/well into a 96-well flat-bottom cell culture plate in the presence or absence of the extracts. Mitochondrial enzyme activity, which is an indirect measure of the number of viable respiring cells, was determined using an MTT reagent after 40 h of treatment with methanol extract or its fractions. Absorbance was read using a microplate reader (Bio-Tek Instruments) at 595 nm. The effect of extracts on cell viability was evaluated as the relative absorbance compared to that of control cultures. LDH leakage is known to be a marker of damage to the cell membrane, and the LDH level was detected using the culture supernatants according to the LDH cytotoxicity detection kit instructions (Takara Shuzo Co.). Cytotoxicity was expressed as the percentage of released LDH (LDH released into the medium/maximal LDH release 100). Maximal

The amount of nitrite was determined using a colorimetric assay (Green et al., 1982). Briefly, 100 μl of cell culture medium was mixed with an equal volume of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine in 5% phosphoric acid) and incubated at room temperature for 10 min. The absorbance at 540 nm was recorded using a microplate reader. Nitrite concentration was determined using extrapolation from a sodium nitrite standard curve. Concentration of prostaglandin E2 (PGE2) in the culture medium was

tested samples, respectively.

**2.3.4 Determination of XOD-inhibitory activity**

sample and the tested samples, respectively.

**2.5 Cytotoxicity assay - MTT and LDH release assays**

**2.6 Measurement of NO and PGE2 production**

LDH release was measured after lysis of the cells with 0.5% Triton X-100.

**2.4 Cell culture**

37°C in a 5% CO2 incubator.

Cells were washed twice with ice-cold PBS and collected. The cells were then treated with a lysis buffer [1× RIPA (Upstate Cell Signaling Solutions, Lake Placid, NY, USA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na3VO4, 1 mM NaF, 1 μg/ml aprotinin, 1 μg/ml pepstatin, and 1 μg/ml leupeptin] and incubated on ice for 1 h. Cell debris was removed by centrifugation and then protein concentration was determined using a Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Cell lysates were subjected to 7.5% or 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with a solution of 0.1% Tween 20/Tris-buffed saline containing 5% nonfat milk powder for 1 h at room temperature. After incubation overnight at 4°C with the indicated primary antibody, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Immunodetection was carried out using an ECL Western blotting detection reagent. The signal intensity of relative bands was determined using image acquisition and analysis software (LabWorks, Cambridge, UK).

#### **2.9 RNA preparation and reverse transcription-polymerase chain reaction (RT-PCR)**

Total cellular RNA was isolated using a Trizol reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's instructions. RNA concentration and purity were determined by measuring absorbance at 260 and 280 nm. A cDNA synthesis was performed using the ImProm-II™ Reverse Transcription System (Promega, Madison, WI, USA) with an oligo dT-15 primer, as recommended by the supplier. PCR analyses were then performed on aliquots of cDNA preparations using a thermal cycler. The reactions were carried out in a 25 l volume containing (final concentration) 1 unit of Taq DNA polymerase, 0.2 mM dNTP, 10 reaction buffer, and 100 pmol primers (Table 1). The cycle number was optimized to ensure product accumulation in the exponential range. -Actin was used as an internal control to normalize the RNA content of each sample. Amplification was initiated at 94°C for 5 min, followed by 18–27 cycles of denaturation at 94°C for 45 s, annealing at the appropriate primer-pair annealing temperature for 45 s, and extension at 72°C for 1 min, followed by a final extension of 10 min at 72°C. After amplification, portions of the PCR reactions were electrophoresed on 2% agarose gel and visualized using ethidium

Antioxidant and Anti-Inflammatory Activities of *Sasa quelpaertensis* Leaf Extracts 141

DPPH radical scavenging activity of the BuOH fraction was more potent than that of other tested samples. For NO scavenging activity, the IC50 value (259.4 μg/ml) was calculated only for the EtOAc fraction; other tested samples exhibited minimal NO scavenging activity. All tested samples (except the water fraction) exhibited XOD-inhibition activity and superoxide radical scavenging activity. XOD-inhibition activity occurred in the following (decreasing) order: EtOAc fraction > hexane fraction > MeOH extract > BuOH fraction. Superoxide radical scavenging activity occurred in the following (decreasing) order: EtOAc fraction > BuOH fraction > hexane fraction > MeOH extract > water fraction. The IC50 values for the EtOAc fraction on XOD-inhibition activity and superoxide radical scavenging activity assay were 32.4 and 21.9 μg/ml, respectively. As shown in Table 2, overall antioxidant activity was more potent in the EtOAc fraction than in any other tested samples.

> Nitric oxide scavenging activity

Methanol extract 862.5 ± 6.4 \* 352.9 ± 16.0 113.5 ± 13.4 Hexane fraction \* \* 238.4 ± 5.6 62.8 ± 4.3 EtOAc fraction 288.9 ± 12.7 259.4 ± 1.6 32.4 ± 1.6 21.9 ± 5.4 BuOH fraction 166.4 ± 9.4 \* 473.5 ± 15.4 23.4 ± 5.9 Water fraction \* \* - 305.2 ± 6.9 Trolox 3.49 ± 0.3 \* 220.3 ± 12.3 32.5 ± 1.9 BHA b) 7.6 ± 0.2 \* 801.7 ± 11.7 \* Allopurinol N/A N/A 1.33 ± 0.1 5.1 ± 0.1 SOD c) N/A N/A \* 4.9 ± 1.9

a) IC50 values were calculated from regression lines using five different concentrations in triplicate

Table 2. Antioxidant activities of the methanol extract and its various fractions from *Sasa*

MeOH extract >2000 1341.7 4.9 >1.8 Hexane fraction 240.1 19.8 175.0 9.6 1.4 EtOAc fraction 465.2 10.2 68.6 0.1 6.8 BuOH fraction >2000 >2000 - Water fraction >2000 >2000 -

b) IC50 is the concentration producing 50% inhibition of NO production in RAW 264.7 cells.

Table 3. Cell toxicity and the effects of the methanol extract and its fractions of Sasa

Sample TC50a) (g/ml) IC50b) (g/ml) Selectivity indexc)

a) TC50 is the concentration producing 50% toxicity in RAW 264.7 cells.

quelpaetensis on LPS-induced NO production in RAW 264.7 cells.

IC50 (g/ml) a)

Uric acid generation activity

Superoxide generation activity

Sample

experiments.

*quelplaertensis* leaf.

b) Butylated hydroxyl anisole c) Superoxide dismutase N/A: Not assay

\* : Can't calculate the value of IC50

c) Selectivity Index = TC50 / IC50.

DPPH radical scavenging activity

bromide staining and a UV transilluminator (SLB Mylmagger; UVP Inc., Upland, CA, USA). The signal intensity of relative bands was determined using image acquisition and analysis software (LabWorks).

Table 1. The sequences of primers used in RT-PCR analysis.

#### **2.10 Transient transfection and luciferase assay**

Cells were seeded at a density of 5 × 103 cells/well into a 96-well flat-bottom cell culture plate and cultured for 18 h. RAW 264.7 cells were transiently transfected with or without a NF-B-promoted luciferase reporter gene plasmid pNF-B-Luc (Promega) and a Renilla luciferase reporter plasmid pRL-TK (Stratagene, La Jolla, CA, USA), to control for transfection efficiency using a FuGENE 6 transfection reagent (Roche, Indianapolis, IN, USA). At 24 h after the start of transfection, cells were incubated with LPS (100 ng/ml) in the presence or absence of the extracts for 24 h. Luciferase activity in the cell lysate was measured using a Dual-Luciferase Reporter Assay Kit (Promega) and a FLUOstar Optima (BMG Labtech, Offenburg, Germany). Luciferase activity was normalized to transfection efficiency, as monitored using a Renilla luciferase expression vector. The level of induced luciferase activity was determined as a ratio to the luciferase activity of unstimulated cells.

#### **2.11 Statistical analysis**

All experiments were conducted in triplicate, but only data from one representative trial are presented. Results are expressed as the mean ± standard deviation (SD). Treatment effects were analyzed using a paired *t*-test.

#### **3. Results**

#### **3.1 Antioxidant activities of** *S. quelpaertensis* **leaf extracts**

Table 2 summarizes the antioxidant activities of *S. quelpaertensis* leaf methanol extract and its fractions, represented by IC50. The IC50 values for DPPH radical scavenging activity against MeOH extract, EtOAc, and BuOH fractions were 862.5, 288.9, and 166.4 μg/ml, respectively. DPPH radical scavenging activity of the BuOH fraction was more potent than that of other tested samples. For NO scavenging activity, the IC50 value (259.4 μg/ml) was calculated only for the EtOAc fraction; other tested samples exhibited minimal NO scavenging activity. All tested samples (except the water fraction) exhibited XOD-inhibition activity and superoxide radical scavenging activity. XOD-inhibition activity occurred in the following (decreasing) order: EtOAc fraction > hexane fraction > MeOH extract > BuOH fraction. Superoxide radical scavenging activity occurred in the following (decreasing) order: EtOAc fraction > BuOH fraction > hexane fraction > MeOH extract > water fraction. The IC50 values for the EtOAc fraction on XOD-inhibition activity and superoxide radical scavenging activity assay were 32.4 and 21.9 μg/ml, respectively. As shown in Table 2, overall antioxidant activity was more potent in the EtOAc fraction than in any other tested samples.

a) IC50 values were calculated from regression lines using five different concentrations in triplicate experiments.

b) Butylated hydroxyl anisole

c) Superoxide dismutase

N/A: Not assay

Phytochemicals as Nutraceuticals 140 – Global Approaches to Their Role in Nutrition and Health

bromide staining and a UV transilluminator (SLB Mylmagger; UVP Inc., Upland, CA, USA). The signal intensity of relative bands was determined using image acquisition and analysis

iNOS Forward 5'-CCCTTCCGAAGTTTCTGGCAGCAGC-3'

COX-2 Forward 5'-CACTACATCCTGACCCACTT-3'

IL-1 Forward 5'-CAGGATGAGGACATGAGCACC-3'

IL-6 Forward 5'-GTACTCCAGAAGACCAGAGG-3'