Antioxidative Activity of Sixty Plants from Iran

Authors

Abstract

Antioxidants are vital substances which possess the ability to protect the body from damage caused by free radical-induced oxidative stress. A variety of free radical scavenging antioxidants exist within the body which many of them are derived from dietary sources like fruits, vegetables and teas.

This article describes a test method for screening the antioxidant activity of 60 Iranian plants of Iran by linoleic acid peroxidation test using 1, 3-diethyl-2-thiobarbituric acid as the reagent. Some plants including Achillea wilhelmsii, Berberis crataegina, Buxus hyrcana, Chrysanthemum cinerariaefolium, Colutea persica, Hyoscyamus niger, Mentha pulegium, Nerium oleander, Pteropyrum aucheri, Rhus coriaria, Rosa canina, Scutellaria pinnatifida, Thymus pubescens, Verbascum alceoides and Ziziphora clinopodioides subsp. rigida showed antioxidant activity (0.41

Keywords


Antioxidative Activity of Sixty Plants from Iran

Iranian Journal of Pharmaceutical Research (2004) 3: 55-59
Received: December 2003
Accepted: February 2004

Original Article

Antioxidative Activity of Sixty Plants from Iran

Effat Souria, Gholamreza Aminb, Anahita Dehmobed-Sharifabadic, Atefeh Nazifia, Hassan Farsama*

 

aDepartment of? Medicinal? chemistry, Faculty? of? Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. bDepartment of Pharmacognosy, Faculty? of? Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. cDepartment of Pharmacognosy, School of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran

 

* Corresponding author: farsam@ams.ac.ir

 

Abstract

 

Antioxidants are vital substances which possess the ability to protect the body from damage caused by free radical-induced oxidative stress. A variety of free radical scavenging antioxidants exist within the body which many of them are derived from dietary sources like fruits, vegetables and teas.

This article describes a test method for screening the antioxidant activity of 60 Iranian plants of Iran by linoleic acid peroxidation test using 1, 3-diethyl-2-thiobarbituric acid as the reagent. Some plants including Achillea wilhelmsii, Berberis crataegina, Buxus hyrcana, Chrysanthemum cinerariaefolium, Colutea persica, Hyoscyamus niger, Mentha pulegium, Nerium oleander, Pteropyrum aucheri, Rhus coriaria, Rosa canina, Scutellaria pinnatifida, Thymus pubescens, Verbascum alceoides and Ziziphora clinopodioides subsp. rigida showed antioxidant activity (0.41<IC50<1.64 mg) comparable to a-tocopherol (IC50=0.60 mg), which was used as the positive control.

 

Keywords: Plant; Antioxidant; Linoleic acid; 1, 3-Diethyl-2-thiobarbituric acid.

 

Introduction

In recent years, it has been established that free radicals and oxidative stress are involved in the pathophysiology of a variety of disorders including atherosclerosis, chronic renal failure, diabetes mellitus, cancer, immune dysfunction and aging (1-6). In relation to these findings an extensive range of antioxidants both exogenous and endogenous, whether synthetic or natural have been presented for the treatment or prophylaxis of disorders attributed to free radical oxidative damages (3, 4, 7). Restriction on the use of synthetic antioxidants due to their probable side-effects has increased the contribution of natural antioxidants (8).

The antioxidant activity of several plant constituents, beyond the vitamins, in the form of crude extract or isolated compound has been put widely into consideration (8-10). Antioxidant activity of many phenolic compounds, including flavonoids, has attracted considerable attention and reported to be more powerful antioxidants than vitamins C, E and b-carotene which are largely in routine use (11). Consumption of the flavonoids and their potential significance as antagonists of oxidative stress has been the interesting subject of many investigations (8, 9, 11, 12). Vegetables and fruits are also reported to decrease the risk of degenerative diseases and could have a protective effect against oxidative stress (11). Antioxidants are also important for food protection against deterioration reactions caused by atmospheric oxygen (8). Considerable effort has been directed in search for safe antioxidants from natural sources. Naturally occurring antioxidants could be found in fruits, vegetables, nuts, seeds, leaves, flowers, roots and barks. Many investigators have found different types of antioxidants in various kinds of plants (8-12).

One of the best approaches for discovering new antioxidants is the screening of plant extracts. This study was carried out as part of a project to investigate the antioxidant activity of 60 selected plants growing in Iran, against linoleic acid peroxidation.

Materials and Methods

Plant material

The plants were collected from different regions of Iran. Information regarding the collection of plants is mentioned in table 1. Voucher specimens of all plants were deposited at the Herbarium of the Faculty of Pharmacy, Tehran University of Medical Sciences. The aerial parts were separated, air dried in the shade, powdered and kept in tightened light-protected containers.

 Chemicals

Linoleic acid, 1, 3-diethyl-2-thiobarbituric acid (DETBA) and quercetin dihydrate were obtained from Merck (Darmstadt, Germany), Aldrich Chemical Co. (Milwaukee, WI, USA) and Fluka Chemical Co. (Buchs, Switzerland) respectively. a-Tocopherol, sodium dodecyl sulfate (SDS) and butylated hydroxytoluene (BHT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and solvents were of analytical grade from Merck.

Extraction

A quantity (50 g) of each powdered plant was extracted in a Soxhlet apparatus with 80% methanol. The methanolic extracts were filtered and evaporated to dryness under reduced pressure in a rotary evaporator. The extracts then transferred to vials, kept at 4?C and examined for antioxidant activity.

Measurment of antioxidant activity

The potential of plant extracts to inhibit peroxidation of linoleic acid was assessed based on a procedure described by Furuta et al. (13). a-Tocopherol was used as the reference compound. For a typical assay three dilutions of each extract (0.02, 0.2 and 2 mg/mL) were prepared. An aliquot of 20 mL of each dilution (equal to 0.4, 4 and 40 mg of extract) was mixed with 20 mL of linoleic acid (2 mg/mL in ethanol) and incubated at 80?C for 60 min. Incubated samples were cooled in an ice bath, followed by the addition of 200 mL of 20 mM BHT, 200 mL of 8% SDS and 400 mL of distilled water. After mixing, 3.2 mL of 12.5 mM DETBA in sodium phosphate buffer (0.125 M, pH 3.0) (warmed to 50?C) was added, mixed and heated at 95?C for 15 min. The mixture was cooled in an ice bath, 4 mL of ethyl acetate was added to each tube, vortexed to extract the pink adduct from the aqueous phase and centrifuged at 1500 rpm for 10 min (F1). A control containing linoleic acid and other additives without antioxidants, representing 100% lipid peroxidation, was also prepared (F2). The blank F1 and F2 solutions were prepared as described above but without linoleic acid. The fluorescence intensity of F1 and F2 samples was measured against their blanks (F3 and F4 respectively) at an excitation wavelength of 515 nm and an emission wavelength of 555 nm in a spectrofluorimeter (Model RF-5000, Schimadzu, Kyoto, Japan). The antioxidant activity was calculated as the percentage of peroxidation inhibition using the following equation (14):

% of peroxidation inhibition= [1-(F1-F3)/(F2-F4)]?100

(equation 1)

 All extracts and the reference substance were assayed at least at three concentrations in triplicates and the results were averaged. A percentage inhibition vs log concentration curve was drawn and the concentration of sample which is required for 50 % inhibition was determined by linear interpolation and expressed as the IC50 value.

Results and Discussion

 

The botanical characteristics of studied plants and inhibitory effect of their methanolic extracts on linoleic acid peroxidation are provided in table 1 in alphabetical order. As displayed in table 1, most plant extracts (44 out of 60) showed more than 80% peroxidation inhibition, using 40 mg of plant extract in the reaction mixture. Plant extracts including Achillea wilhelmsii, Buxux hyrcana, Chrysanthemum cinerariaefolium, Colutea persica, Hyoscyamus niger, Mentha pulegium, Myrtus communis, Nerium oleander, Paliurus spina-christi, Peganum harmala, Pterocarya fraxinifolia, Rhus coriaria, Rosa canina, Smilax excelsa, Thymus migricus, Thymus pubescens, Verbascum alceoides and Ziziphora clinopodioides subsp. rigida showed more than 70% inhibition, using 4 mg of each plant extract. A limited number of plant extracts including Berberis crataegina, Colutea persica, Mentha pulegium, Pteropyrum aucheri and Rosa canina showed more than 40% inhibition, using 0.4 mg of plant extract in the reaction mixture. In all cases the antioxidant activity increased with increasing the concentration. IC50 values of the studied plants showed considerable differences with each other in the range of 0.41-45.76 mg. IC50 values of some plant extracts including Chrysanthemum cinerariaefolium (0.57 mg), Colutea persica (0.41 mg), Mentha pulegium (0.57 mg) and Rosa canina (0.41 mg) was lower then a-tocopherol (IC50= 0.60 mg). The IC50 values of Berberis crataegina (0.81 mg), Buxus hyrcana (1.15 mg), , Hyoscyamus niger (0.64 mg), Pteropyrum aucheri (0.94mg), Rhus coriaria (0.91mg), Scutellaria pinnatifida (0.76 mg), Thymus pubescens (0.84 mg) and Verbascum alceoides (0.87 mg) were within the range of? 0.64-1.15 mg, which is approximately in the range of? a-tocopherol (IC50= 0.60 mg).

 

Table 1. The botanical names and antioxidant activities of aerial parts of 60 plant extracts from Iran

 

 

 

 

 

 

 

Peroxidation inhibition(%)

 

No.

Location

Date*

Alt*

Voucher No.

Scientific name

Family name

0.4 μg

4 μg

40 μg

IC50 (μg)

1

N. of Karadj

99/6/6

1300

6570-TEH

Achillea wilhelmsii C. Koch

Compositae

27.37?2.66

78.93?0.65

75.27?4.05

1.41? 0.27

2

E. of? Karadj

99/6/9

1200

6586-TEH

Achillea tenuifolia Lam.

Compositae

28.09?4.19

60.57?3.20

88.06?3.14

1.88? 0.37

3

E. of Karadj

99/6/9

1120

6585-TEH

Acroptilon repens (L.) DC. subsp. repens

Compositae

26.72?5.06

46.85?0.39

85.88?1.82

4.68? 0.13

4

Vaysar, S. of Chalus

99/6/4

600

6567-TEH

Asperula stylosa Trin

Rubiaceae

15.56?4.20

48.38?2.15

82.65?3.29

4.51? 0.57

5

E. of Tehran

99/7/21

1100

6587-TEH

Astrodaucus orientalis (L.) Drude

Umbelliferae

22.28?2.65

58.09?1.63

83.11?2.82

2.86? 0.27

6

Margun, W. of Shiraz

99/7/22

2000

6619-TEH

Ballota aucheri Boiss

Labiatae

2.65?1.63

47.92?1.86

90.54?4.57

5.09? 0.88

7

Ruin, Khorassan

99/6/12

1500

6590-TEH

Berberis crataegina DC.

Berberidaceae

49.95?4.65

52.02?3.07

90.29?2.18

0.81? 0.30

8

Talesh N. of Iran

99/6/8

930

6582-TEH

Buxus hyrcana Pojark

Buxaceae

35.23?6.83

71.92?8.31

86.94?2.79

1.15? 0.45

9

W. of Karadj

99/6/4

1100

6561-TEH

Capparis spinosa L.

Capparidaceae

20.61?0.69

49.29?6.78

64.58?6.83

5.84? 2.85

10

E. of Tehran

99/6/21

1100

6596-TEH

Carthamus oxyacantha M.B.

Compositae

27.05?0.15

28.35?2.18

71.27?2.50

9.11? 0.92

11

Ruin, Khorassan

99/6/12

1500

6616-TEH

Centaurea bruguieriana (DC.) Hand.-Mzt. subsp. belangerana (DC.) Bornm.

Compositae

25.63?7.44

62.98?0.37

91.00?5.65

1.74?0.24

12

E. of Tehran

99/6/21

1100

6598-TEH

Chenopodium botrys L.

Chenopodiaceae

31.72?1.05

36.26?4.07

51.59?2.57

45.76?14.4

13

Km.40 Lowshan ? Rasht

99/6/8

1350

6583-TEH

Chrysanthemum cinerariaefolium ( Trev ) Vis.

Compositae

37.52?2.02

86.96?1.45

85.20?3.56

0.57?0.11

14

E. of? Marzanabad

99/6/4

850

6566-TEH

Colutea persica Boiss.

Leguminosae

46.01?4.93

76.32?4.35

81.52?3.67

0.41?0.11

15

E. of Tehran

99/6/21

1200

6597-TEH

Erodium oxyrrhynchum M.B. subsp. Oxyrrhynchum

Geraniaceae

22.85?0.42

41.51?0.89

71.00?2.68

6.57?1.04

16

Near Shush

99/7/21

850

6618-TEH

Eucalyptus camaldulensis Dehn.

Myrtaceae

19.38?0.31

62.99?0.55

92.88?1.38

2.16?0.10

17

Hezarcham, Chalus-Karadj

99/6/22

2500

6613-TEH

Ficus carica L. subsp. Carica

Moraceae

27.12?6.14

52.53?0.99

97.05?1.62

3.18?0.33

18

Loshan, Ghazvin-Rasht

99/6/8

1030

6589-TEH

Glaucium contortuplicatum Boiss.

Papaveraceae

32.16?6.43

51.39?7.83

55.18?2.41

8.68?2.63

19

Aderan N. of Karadj

99/6/6

1300

6579-TEH

Glaucium elegans Fisch.& C.A.Mey.

Papaveraceae

7.79?1.25

38.46?0.27

61.76?1.23

14.20?0.61

20

Kandowan N. Karadj

99/6/6

2600

6578-TEH

Glaucium fimbrilligerum Boiss.

Papaveraceae

36.26?4.28

48.63?2.61

89.16?8.01

4.19?0.78

21

Ruin , Khorassan

99/6/12

1500

6593-TEH

Glycyrrhiza glabra L. var glabra

Leguminosae

0.86?1.29

62.19?1.59

96.14?0.71

2.86?0.70

22

Kandowan, N. of Karadj

99/6/22

2200

6612-TEH

Hyoscyamus niger L.

Solanaceae

22.96?7.64

71.86?1.03

91.19?5.08

1.64?0.32

23

Vaysar, S. of Chalus

99/6/4

600

6569-TEH

Hypericum androsaemum L.

Hypericaceae

22.84?2.43

55.37?4.55

93.37?2.34

2.60?1.13

24

Ruin, Khorassan

99/6/12

1500

6594-TEH

Linaria pyramidata (Lam.) Sprengl

Scrophullariaceae

18.78?1.68

39.60?2.42

79.31?3.08

5.57?0.93

25

Marzanabad, Chalus

99/6/4

650

6563-TEH

Marrubium vulgare L.

Labiatae

30.35?1.03

41.58?6.16

89.51?1.12

5.61?1.17

26

Rasht

99/6/9

350

6580-TEH

Mentha pulegium L.

Labiatae

45.64?6.95

81.86?0.93

81.44?2.51

0.57?0.29

27

Kandowan

99/6/22

2700

6610-TEH

Minuartia lineata Bornm.

Caryophyllaceae

38.99?3.38

50.48?5.91

96.16?1.50

3.34?1.55

28

Gachsaran- Shiraz

99/7/21

1200

6623-TEH

Myrtus communis L.

Myrtaceae

11.56?4.26

78.06?1.38

90.57?2.45

2.40?0.49

29

Polur, Haraz ? Amol

99/6/21

1800

6604-TEH

Nepeta glomerulosa Boiss. subsp. glomerulosa

Labiatae

15.77?1.97

45.11?4.66

94.78?0.90

4.40?1.16

30

Tehran

99/6/17

1100

6615-TEH

Nerium oleander L.

Apocyanaceae

19.05?0.94

80.95?6.43

92.56?1.37

1.51?0.29

31

Yasouj ? Esphahan

99/7/22

1200

6625-TEH

Ononis spinosa L.

Leguminosae

15.83?3.17

68.00?0.55

91.06?1.50

2.37?0.36

32

Marzanabad, Chalus

99/6/4

650

6564-TEH

Paliurus spina ? christi Miller

Rhamnaceae

19.07?8.39

82.49?3.79

85.38?2.91

1.83?0.97

33

Polur, Haraz - Amol

99/6/21

1800

6603-TEH

Papaver bracteatum Lindl.

Papaveraceae

32.65?3.01

51.05?0.26

92.21?1.67

3.51?0.10

34

Aderan N. of Karadj

99/6/8

1300

6572-TEH

Peganum harmala L.

Zygophylaceae

10.20?3.16

71.51?3.38

70.15?5.07

1.98?0.13

35

Kandowan

99/6/22

2700

6608-TEH

Phlomis anisodonta Boiss.

Labiatae

33.03?1.43

48.73?4.53

88.34?8.91

3.14?1.16

36

Nowshahr

99/6/22

10

6627-TEH

Phytolacca americana L.

Phytolaccaceae

31.51?6.00

65.84?3.60

68.77?0.16

2.08?0.56

37

Gachsaran- Shiraz

99/7/21

1200

6624-TEH

Pistacia atlantica Desf. subsp.mutica? ????????????????(Fisch. &? C.A. Mey.) Rech. f.

Anacardiaceae

28.75?7.13

50.86?3.93

56.46?2.79

7.51?6.14

38

E. of Tehran

99/6/21

1200

6599-TEH

Prosopis stephaniana (M.B )Kunth. ex spreng

Anacardiaceae

38.01?1.60

36.07?1.43

57.37?2.04

18.61?2.83

39

Asalem

99/7/14

150

6626-TEH

Pterocarya fraxinifolia (Poir.) Spach

Juglandaceae

5.94?4.39

73.87?1.58

93.99?2.68

2.60?0.11

40

Ghazvin

99/6/19

1200

6588-TEH

Pteropyrum aucheri Jaub.&. Spach

Polygonaceae

41.34?1.51

63.46?2.63

89.82?1.16

0.94?0.13

41

N. of Shiraz

99/7/20

2900

6621-TEH

Rhamnus cornifolia Boiss. & Hohen. subsp.cornifolia

Rhamnaceae

22.38?2.45

61.93?3.05

88.40?2.27

2.27?0.42

42

N. of? Karadj

99/6/22

300

6614-TEH

Rhus coriaria L.

Anacardiaceae

33.45?1.72

77.82?1.40

93.81?2.13

0.91?0.04

43

Ruin, Khorassan

99/6/12

1500

6591-TEH

Roemeria refracta DC.

Papaveraceae

14.13?0.50

54.89?2.62

93.96?1.09

2.79?0.14

44

S. of Chalus

99/6/6

2100

6576-TEH

Rosa canina L.

Rosaceae

49.52?8.91

77.63?7.05

91.79?3.56

0.41?0.18

45

Tehran ? Amol

99/6/21

2500

6601-TEH

Salvia hypoleuca Benth.

Labiatae

9.95?4.88

42.05?0.17

91.90?1.55

5.27?0.10

46

Ruin, Khorassan

99/6/12

1500

6592-TEH

Salvia macrosiphon Boiss.

Labiatae

24.20?9.90

58.50?6.59

91.52?0.20

2.96?0.91

47

Tehran ? Amol

99/6/21

2000

6574-TEH

Salvia verticillata L.

Labiatae

38.67?3.14

42.70?2.20

76.01?1.84

6.38?0.55

48

E. of Tehran

99/6/21

1100

6595-TEH

Scariola orientalis (Boiss.) Sojak

Compositae

18.86?3.04

57.08?1.00

71.24?7.77

2.71?0.20

49

N. Shiraz

99/7/20

3200

6620-TEH

Scutellaria multicaulis Boiss. subsp. multicaulis

Labiatae

18.44?0.29

49.16?7.41

92.48?1.13

2.83?0.24

50

Kandowan

99/6/22

2700

6611-TEH

Scutellaria pinnatifida Art.et Hamilt.

Labiatae

47.60?5.88

64.06?7.91

94.87?2.19

0.76?0.27

51

E. of Assalem

99/6/12

250

6584-TEH

Senecio cineraria DC.

Compositae

36.31?3.63

37.55?2.07

83.26?6.14

7.29?1.41

52

E. of Tehran

99/6/21

1100

6633-TEH

Silene coronaria (L.) Clairv

Caryophyllaceae

22.65?2.59

64.92?6.82

89.11?1.54

2.04?0.42

53

Assalem

99/7/14

150

6628-TEH

Smilax excelsa L.

Asparaginaceae

13.19?2.17

78.10?5.12

94.21?0.70

2.00?0.36

54

NE. of Tehran

99/6/21

2100

6600-TEH

Sophora alopecuroides L.

Leguminosae

12.32?2.32

45.20?0.48

94.09?2.81

4.70?0.10

55

Kandowan

99/6/22

2700

6607-TEH

Stachys lavandulifolia Vahl.

Labiatae

23.76?0.42

52.25?5.74

91.56?2.16

3.11?1.15

56

Lahijan

99/6/21

30

6602-TEH

Tamarix aralensis Bge

Tamaricaceae

4.59?5.29

17.62?2.66

54.30?4.07

34.70?7.86

57

Kandowan

99/6/22

2700

6609-TEH

Thymus migricus klokov.& Desj.- Shost.

Labiatae

37.94?6.05

81.17?2.46

97.00?1.56

0.77?0.06

58

NE. of Tehran

99/6/21

2100

6605-TEH

Thymus pubescens Boiss. & Kotschy ex Celak.

Labiatae

38.86?1.49

72.68?0.55

97.42?1.12

0.84?0.03

59

Vaysar, S. of Chalus

99/6/4

600

6568-TEH

Verbascum alceoides Boiss. &. Hausskn.

Scrophulariaceae

37.82?0.83

72.79?6.97

75.99?3.30

0.87?0.021

60

Taleghan

99/7/21

1400

6617-TEH

Ziziphora clinopodioides Lam. subsp. rigida (Boiss.) Rech. f.

Labiatae

27.21?4.16

70.99?4.73

91.73?0.93

1.45?0.31

* Date=Date of collection; Alt=Altitude (m)

 

Presence of unsaturated fatty acids in the lipid membranes, especially linoleic acid, makes them very susceptible to oxidative reactions. Inhibition of linoleic acid oxidation could be a good indication for antioxidant activity and has been widely used. In this study methanolic extracts of 60 plant species of Iran were evaluated for their antioxidant activity within at the range of 0.4 to 40 mg of the plant extracts against 4 mg of linoleic acid peroxidation in the reaction mixture. Linoleic acid peroxidation was determined spectrofuorimetrically, using 1, 3-diethyl-2-thiobarbituric acid as the reagent.

Natural antioxidants are usually phenolic and polyphenolic (including flavonoids) compounds (8, 15). The presence of these compounds in several plants examined in this study has already been reported as mentioned below:

Achillea wilhelmsii (16, 17), Buxus hyrcana (18), Eucalyptus camaldulensis (19, 20),? Mentha pulegium (21), Myrtus communis (22-24), Nerium oleander (25), Paliurus spina-christi (26, 27), Peganum harmala (28), Rhus coriaria (29), Rosa canina (30, 31), Senecio cineraria (32), Sophora alopecuroides (33) and Ziziphora clinopodioides (34).

?There are numerous of reports stating that the risk of degenerative diseases is diminished in people consuming large quantities of vegetables and fruits (11, 35, 36). At the same time it should be taken in to account that the antioxidant defense system of the human body is composed of different antioxidant compounds (12). The quality and antioxidant capacity of vegetables have also been recognized as effective supplement (11). Thus, the plants investigated in this study could provide protection against oxidative stress. However it is not known that whether components of the extracts are responsible. Further studies are in progress to elucidate identity of responsible compounds.

Acknowledgement

The authors would like to thank the research councils of Tehran University of Medical Sciences, and the Iranian Ministry of Health and Education for the financial support of this research project.

References 

(1)     Noguchi N and Niki E. Phenolic antioxidants: A rationale for design and evaluation of novel antioxidant drug for atherosclerosis. Free Rad. Biol. Med. (2000) 28: 1538-46

(2)     Young IS and Woodside JV. Antioxidants in health and disease. J. Clin. Pathol. (2001) 54: 176-86

(3)     Halliwell B. The antioxidant paradox. Lancet (2000) 355: 1179-80

(4)     Maxwell SRJ. Prospects for the use of antioxidant therapies. Drugs (1995) 49: 345-61

(5)     Heinecke JW. Oxidative stress: new approaches to diagnosis and prognosis in atherosclerosis. Am. J. Cardiol. (2003) 91: 12A-16A

(6)     Metodiewa D and Koska C. Reactive oxygen species and reactive nitrogen species: relevance to cyto(neuro)toxic events and neurologic disorders. An overview. Neurotox. Res. (2000) 1: 197-233

(7)     Cesquini M, Torsoni MA, Stoppa GR and Ogo SH. t-BOOH-induced oxidative damage in sickle red blood cells and the role of flavonoids. Biomed. Pharmacother. (2003) 57: 124-9

(8)     Velioglu YS, Mazza G, Gao L and Oomah BD. Antioxidant activity and total phenolics in selected fruits, vegetables and grain products. J. Agric. Food Chem. (1998) 46: 4113-17

(9)     Gazzani G, Papetti A, Massolini G and Daglia M. Anti and prooxidant activity of water soluble components of some common diet vegetables and the effect of thermal treatment. J. Agric. Food Chem. (1998) 46: 4118-22

(10)  Larson RA. The antioxidants of higher plants. Phytochem. (1988) 27: 969-78

(11)  Vinson JA, Hao Y, Su X and Zubik L. Phenol antioxidant quantity and quality in foods: vegetables. J. Agric. Food Chem. (1998) 46: 3630-34

(12)  Cao G, Sofic E and Pior RL. Antioxidant capacity of tea and common vegetables. J. Agric. Food Chem. (1996) 44: 3426-31

(13)  Furuta S, Nishiba Y and Suda I. Fluorometric assay for screening antioxidative activity of vegetables. J. Food Sci. (1997) 62: 526-28

(14)  ?Okada Y and Okada M. Scavenging effect of soluble proteins in broad beans on free radicals and active oxygen species. J. Agric. Food Chem. (1998) 46: 401-406

(15)  Kim BJ, Kim JH, Kim HP and Heo MY. Biological screening of 100 plant extracts for cosmetic use (II): antioxidative activity and free radical scavenging activity. Int. J. Cosmetic Sci. (1997) 19: 299-307

(16)  Ahmad AA, El-Sayed NH and Mabry TJ. Flavonoids of Achillea santolina and A. fragrantissima (Compositae, Anthemideae). Rev. Latinoam. Quim. (1989) 20: 5

(17)  Khafagy SM, Sabri NN, Soliman FSG, Abou-Donia AH and Mosandl A. Isolation of two flavonoids from Achillea santolina L. growing in Egypt. Pharmazie (1976) 31: 894-5

(18)  Aynehchi Y, Salehi Sormaghi MH and Amin GR. Survey of Iranian plants for saponins, alkaloids, flavonoids and tannins III. Int. J. Crude Drug Res. (1985) 23: 33-41

(19)  Osawa T and Namiki M. A novel type of antioxidant isolated from leaf wax of Eucalyptus leaves. Agric. Biol. Chem. (1981) 45: 735-9

(20)  Cadahia E, Conde E, Garcia-Vallejo MC and De Simon B. High pressure liquid chromatographic analysis of polyphenols in leaves of Eucalyptus camaldulensis, E. globulus and E. rudis: proanthocyanidins, ellagitannins and flavonol glycosides. Phytochem. Anal. (1997) 8: 78-83

(21)  Zaidi F, Voirin B, Jay M and Viricel MR. Free flavonoid aglycones from leaves of Mentha pulegium and Mentha suaveolens (Labiatae). Phytochem. (1998) 48: 991-4

(22)  Hadafi A, Ismaili Alaoui M, Chaouch A, Benjilali B and Zrira S. Antioxidant activity and phenolic acid content in rosemary (Rosmarinus officinalis L.) and myrtle (Myrtus communis). Riv. Ital. EPPOS? (1998) 325-340

(23)  Hinou J, Lakkas N and Philianos S. Polyphenolic constituents of Myrtus communis. Plant Med. Phytother. (1988) 22: 98-103

(24)  Hinou J, Demetzos C and Harvala C. Polyphenolic compounds of Myrtus communis. Fitoterapia (1989) 60: 94

(25)  Hiermann A, Kartnig T and Lukasczyk K. Occurrence of flavonoids in Nerium oleander. Sci. Pharm. (1982) 50: 71-5

(26)  Kustrak D, Males Z and Pitarevic I. Determination of the content of total polyphenols and tannins of Christ?s thorn (Paliurus spina-christi Mill.). Farm. Glas. (1993) 49: 105-9

(27)  Kustrak D, Males Z and Pitarevic I. Determination of the flavonoid content of Christ?s thorn (Paliurus spina-christi Mill.). Farm. Glas. (1992) 48: 101-6

(28)  Sharaf M, El-Ansari MA, Matlin SA and Saleh NAM. Four flavonoid glycosides from Peganum harmala. Phytochem. (1997) 44: 533-536

(29)  Van Loo P, De Bruyn A and Verzele M. Liquid chromatography and identification of the flavonoids present in the sumac tannic acid extracted from Rhus coriaria. Chromatographia (1988) 25: 15-20

(30)  Costantino L, Rastelli G, Rossi T, Bertoldi M and lbasini A. Composition, superoxide radicals scavenging and antilipoperoxidant activity of some edible fruits. Fitoterapia (1994) 65: 44-7

(31)  Tarnoveanu DS, Rapior S, Gargadennec A and Andary C. Flavonoid glycosides from the leaves of Rosa canina. Fitoterapia (1995) 66: 381-2

(32)  Cezard C, Torck M and Pinkas M. Senecio cineraria DC. preliminary study. Plant Med. Phytother. (1984) 18: 160-4

(33)  Shuyu L, Linia H, Haiyan L, Shumin L and Zhiyan G. Isolation and identification of a new biflavone C-glycoside in fontail-like Sophora (Sophora alopecuroides). Zhongcaoyao (1992) 13: 395-6

(34)  Oganesyan GB, Galstyan AM, Mnatsakayan VA, Paronikyan RV and Ter-Zakharyan YuZ. Phenol and flavonoids of Ziziphora clinopodioides. Khim. Prir. Soedin. (1991) 2: 286-7

(35)  Gillman MW, Cupples A, Gagnon D, Posner BM, Ellison RC, Gastelli WP and Wolf PA. Protective effect of fruits and vegetables on development of stroke in men. J. Am. Med. Assoc. (1995) 273: 1113-1117

(36)  Ames BN, Gold LS and Willett WC. The causes and prevention of cancer. Proc. Natl. Acad. Sci. USA. (1995) 92: 5258-5265