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RESEARCH ARTICLE
Year : 2009  |  Volume : 1  |  Issue : 3  |  Page : 125-129 Table of Contents     

Biological activity of Crambe orientalis L. growing in Iran


1 Department of Biology, University of Mohaghegh Ardabili, Ardabil, Iran
2 Depatrment of Animal Sciences, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran

Date of Submission10-Mar-2009
Date of Acceptance09-Apr-2009
Date of Web Publication2-Jan-2010

Correspondence Address:
S M Razavi
Department of Biology, University of Mohaghegh Ardabili, Ardabil
Iran
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Source of Support: None, Conflict of Interest: None


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   Abstract 

Crambe orientalis (Brassicaceae) is a perennial herb growing wild in Iran. Cytotoxic, antioxidant, antimicrobial and phttotoxic effects of the plant leaves extracts have been evaluated by MTT, DPPH, Disc diffusion and lettuce assays, respectively. Our results showed that dichloromethane and methanol extracts of the plant leaves exhibited high cytotoxic effects against Mc-Coy cell line with IC 50 , value of 659 and 432 μg/ml, respectively. The extracts indicated high antioxidant activity with RC 50 , value of 33.3 and 40.5 μg/ml, respectively. Methanol extract showed strong antibacterial activity against both gram-positive and gram-negative bacteria. All the extracts displayed significant allelopatic potential, as well as. It is assumed that biological activity of the plant may be related to presence of isothiocyanates in the plant leaves.

Keywords: Crambe oriental is, Antioxidant, Cytotoxic, Antimicrobial, Allelopathy


How to cite this article:
Razavi S M, Zarrini G, Zahri S, Ghasemi K, Mohammadi S. Biological activity of Crambe orientalis L. growing in Iran. Phcog Res 2009;1:125-9

How to cite this URL:
Razavi S M, Zarrini G, Zahri S, Ghasemi K, Mohammadi S. Biological activity of Crambe orientalis L. growing in Iran. Phcog Res [serial online] 2009 [cited 2020 Dec 2];1:125-9. Available from: http://www.phcogres.com/text.asp?2009/1/3/125/58122


   Introduction Top


Crambe orientalis L. is a perennial herb growing to 1.2 m in fields, hills, dry slops, rocky places and clay soils. The plant has a wide distribution from Europe, East Mediterranean to west Asia-Iran [1] . It has large lyrate­pinnatifid leaves that may reach to 60 cm in length and have kale-like odor. Sepideh is the common name in Iran where it used as animal fodder. Some species of Crambe may have edible uses in different parts of the world. Young leaves of C. orientalis have a pleasant almost nutty flavor and go well in a mixed salad or also make a very pleasant cooked vegetable in some European countries where it named as oriental sea­kale [2] .

In a previous work, we studied the chemical composition of essential oil of leaves and flowers of C. orientalis. The oils was dominated of isothiocyanates that caused phttotoxic and very strong cytotoxic effects of the oils [3] . In the present work, we investigate some biological effects of the plant extracts.


   Materials and Methods Top


Plant material - Plant materials were collected from Ardabil in north west of Iran. A sample of this plant has been deposited at the herbarium for medicinal plants at the faculty of science of the University of Mohaghegh Ardabili (No:1387-2).

Plant extractions

The plant leaves was soxhlet extracted with n-hexane, dichloromethane and methanol, respectively. The extracts were dried in vacuo.

Cytotoxicity assay

Mc-Coy cell lines (Pasteur, C123) were grown in RPMI 1640(Gibco, No 51800-019) medium. Each 500 ml of the medium supplemented with 10% heat- inactivated fetal calf serum (FCS) in deionized water [4] . The stock solutions of dichloromethane and methanol extracts of C. orientalis were prepared by dissolving the compound in 100 μL dimethylsulphoxide(DMSO). The final concentration of the extract was 1, 0.4, 0.3 0.2 and 0.1 mg/ml. Cells were plated in the appropriate media on 24-well micro plates in a 500 μl total volume at a density of 6x10 5 cell/ml. Triplicate wells were treated with media containing different concentration of the extract. The plates were incubated at 37˚C in 5% C02 for time course of 24 h. Cell viability was evaluated by the MTT colorimetric technique [5] . The OD 570 , was determined using a spectrophotometer. Media- only treated cells served as the indicator of 100% cell viability. The 50% inhibitory concentration (IC 50) was defined as the concentration that reduced the absorbance of the untreated wells by 50% of the control in the MTT assay. Viability percentage was evaluated as OD treatment/ OD control [6] .

Antioxidant assay

Serial dilutions were carried out with the stock solutions (1 mg/ml) of the plant extracts to obtain concentrations 0.5, 0.25, 0.175, 0.087, 0.043, 0.021, 0.010, .005, 0.002 and 0.001 mg/ml. All of the solutions were prepared by methanol as solvent. Diluted solutions (5ml each) were mixed with 5 ml of 2,2- Biphenyl- 1- picryl hydrazyl (DPPH, Sigma) and allowed to stand for 3 min for any reaction to occur. The UV absorbance was recorded at 517 nm. The experiment was performed in duplicate and the average absorption was noted for each concentration. The RC 50 value, which is the concentration of the test material that reduced 50% of free radical concentration, was calculated as mg/ml [7] .

Antimicrobial assay

The antibacterial and antifungal activities of the plant extracts were determined against Bacillus subtilis (PTCC 1207), Bacillus cereus (PTCC 1247), Staphylococcus epiderrnidis (PTCC 1114),  Escherichia More Details coli (PTCC 1047), Candida kefyr (ATCC 1140) and Candida krusei (ATCC 44507) by the disc diffusion method [12] . Muller- Hinton agar (MHA) (oxoid)) and sabouraued dextrose agar (SDA) were used for preparation of the media for bacteria and fungi strains, respectively. The filter paper discs (6mm in diameter) were individually impregnated with 10 pl of stock solution of the extracts (3 mg/ml) and then placed onto the agar plates which had previously been inoculated with the tested microorganisms. The plates were inoculated with bacteria incubated at 37˚C for 24 h and at 30˚C for 48h for fungal strains. The diameters of inhibition zones were measured in millimeters. All the tests were performed in duplicate. Gentamicin (30pg) served as positive control. The MICs of the extracts against the test microorganisms were determined by the Agar dilution method [8] .

Phytotoxic assay

Lettuce (Lactuca sativa L. cv. Varamin) seeds were used to test germination response to different concentration of the plant leaves extracts. Hex and DCM extracts were dispersed as an emulsion in water using Tween 20. Four concentrations of the extracts (0.1, 1, 5 and 10 mg/ml) were obtained by dilution of the emulsions with deionized water The stock solution of Met extract was prepared by sterile waters as and different concentration of extract (0.1, 1, 5 and 10 mg/ml) were obtained by dilution with demonized water. All seeds were surface sterilized with sodium hypo chloride (1%). Four replicates, each of 25 seed, were prepared for each treatment using sterile  Petri dish More Detailses (90mm) lined with one sterile filter paper (Whatman, number 2). 5 ml of different concentration of the extracts was added to each Petri dish. Prepared plates were then placed in a germination cabinet at 25˚C in the dark. After 1 week, in the each treatment, germination percentage was determined, root and shoot length was measured [9] .

Statistical analysis

In all assays, SPSS 11.5 software was used for statistical analysis. Analysis of variance (ANOVA) followed by Duncan test was used to see the different amongst various groups. The significance level was set at p<0. 05.


   Results and Discussion Top


Our results showed that dichloromethane (DCM) and methanol (Met) extracts of the leaves of C. orientates exhibited strong cytotoxic effects against Mc-Coy cell lines, with an IC 50 value of 659 and 432 μmol/ml, respectively. As shown in the [Figure 1], MTT assay showed that the addition of extracts of leaves reduce the viability of Mc-Coy cells in a dose-dependent manner. Following a 24-h incubation with 1 mg/ml of the DCM and Met extracts, viability of cells was observed to be 22.9 and 20.7%, respectively.

The DPPH assay showed that DCM and Met extracts of the plant have high antioxidant activity with RC 50 value of 33.3 and 40.5 μg/ml, respectively. However, the Hex extract exhibited low antioxidant potential with RC 50 value of 1390 μg/ml.

Results of antimicrobial assay are presented in [Table 1]. As it shown, while the Met extract of C. orientates exhibited very high antimicrobial effects, the Hex and DCM extracts showed no antimicrobial activity. The Met extract indicated strong antibacterial activity against all tested bacteria with inhibition zones about (23-34 mm) and MIC value of 250-500 μg/ml. The extract exhibited modest antifungal effects against Candida kefyr and Candida krusei with inhibition zone of 13.9 and 18.5 mm, respectively.

The results of our study showed that all the three extracts of C. orientates, significantly exhibited phytotoxic effects and can reduce seed germination shoot and root growth of lettuce at concentrations higher than 0.1 g/ml [Table 2]. The methanolic extract has strongest effects rather than other extracts.

There are many reports on the isolation of glucosinolates and their degradation products like isothiocyanates from leaves of different species of Crambe [10] . We had also previously identified isothiocyanates in essential oil of C. orientates leaves [3] . It is assumed that isothiocyanates may be responsible for some biological activity of Crambe genus like cytotoxic and phytotoxic effects. There is strong evidence that isothiocyanates inhibit carcinogen ­induced tumorogenesis in some human organs [11] . Thus, high cytotoxic potential of the plant leaves extracts could be due to presence of isothiocyanates. We previously described the high cytotoxic effects of the essential oil of C. orientatis leaves on Mc-Coy cell lines with very low IC 50 value as 16 μg/ml 3 . The comparison of our previous data with present results showed that the essential oil of the plant leaves exhibit more anti proliferative potential than leaves extracts.

Previous phytochemical reports showed that there are different flavonoids like quercetin, apigenin, kaempferol and loteolin in the leaves of Crambe genus [12] . It is assumed that high antioxidant potential of Met and DCM extracts of the plant leaves is dependent to flavonoides.

These results suggest that C. orientates leaves may act as chemotherapeutic and chemopreventive agent against cancer. High consumption of the plant leaves as mixed salad could be associated with a reduced risk of cancer.

On the other hand, our results showed that the Met extract of C. orientates leaves displayed a broad antimicrobial spectrum and exerted strong antimicrobial effect against both gram-positive and gram-negative bacteria. Like some other biological effects, the strong antimicrobial activity of the extract could be due to isothiocyanates that has been reported to occur in the plant [3] . This compound class can easily penetrate biomembranes, hence, they are considered bioactive agent that play a defense role for plant against pathogens and herbivores [13] . Due to high antimicrobial potential of C. orientates leaves, it can be used as antiseptic agent to eliminate antibiotic resistance microorganisms.

The results of lettuce phytotoxic assay showed that all of the three extracts of C. orientates leaves, exhibited modest allelopathic effect. The allelopathy potential of plants has been shown to play important roles in the determination of plant diversity, dominance, succession and climax of natural vegetation and in the plant productivity of agroecosystems [14] . It is supposed that allelopathy potential of the plant might also be attributed to glucosinolates and isothiocyanates. This potential may cause a considerable resistant against weeds, pathogens and herbivores in farming of the plant as a crop.

 
   References Top

1.S. Mobayen, Flora of Iran (Tehran University Publication, Tehran, 1985) pp 584-586.  Back to cited text no. 1      
2.E. Launert, Edible and medicinal plants (Hamlyn Press, London, 1981).  Back to cited text no. 2      
3.S.M. Razavi S. Nejad-Ebrahimi. Chemical composition, allelopathic and cytotoxic effects of essential oils of flowering tops and leaves of Crambe orientalis L. from Iran. Nat. Prod. Res. In Press (2009).  Back to cited text no. 3      
4.Q. Zhang, J. Wu, Z. Hu and D. Li. Induction of HL­60 apoptosis by ethyl acetate extract of Crdyceps sinensis fungal mycelium. Life Sci. 75 (24): 2911-2919 (2004).  Back to cited text no. 4      
5.Doyle and J.B. Griffiths, Cell and tissue culture, Laboratory procedures in biotechnology press, (John whey Press, West Sussex, 1998).  Back to cited text no. 5      
6.Yeldjou, P. Moree and P.B. Techounwou. Dose and time-dependent response of human leukemia (HL-69) cells to Arsenic trioxide treatment. Int. J. Environ. Res. Public Health 3(2): 136-140(2006).  Back to cited text no. 6      
7.S.M. Razavi, H. Nazemiyeh, R. Hajiboland, Y. Kumaramasamy, A. Delazar and L. Nahar. Coumarins from aerial parts of Prangos uloptera. Braz. J. Pharmacogn. 18 (1): 1-5 (2008).  Back to cited text no. 7      
8.V. Lorain, Antibiotic in Laboratory Medicine (Williams & Willkins Publication, Philadelpia,1996).  Back to cited text no. 8      
9.L.V. Jefferson and M. Pennacchio. Allelopathic effects of foliage extracts from four Chenopodiaceae species on seed germination. J. Arid Environ. 55(2): 275-285 (2003).  Back to cited text no. 9      
10.Quinsac, A. Charrier, D. Ribaillier and J.Y. Peron. Glucosinolates in etiolated sprouts of sea-kale (Crambe martima L.). J. Scien. Food and Agri. 65 (2): 201-207 (2006).  Back to cited text no. 10      
11.L. Tang and Z. Yuesheng. Dietry isothiocyanates inhibit the growth of human bladder carcinoma cells. J. Nutrit. 134(8): 2004-2010 (2004).  Back to cited text no. 11      
12.J. Onyilagha, A. Bala, R. Hallett, M. Gruber, J. Soroka and N. Westcott. Leaf flavonoids of the cruciferous species, Camelina saliva, Crambe Spp., Tblaspi arvense and several other genera of the family Brassicaceae. Bichem. Syst. Ecol. 31(11): 1309-1322 (2003).  Back to cited text no. 12      
13.J.T. Romeo, Integrative phytochemistry: from ethnobotany to molecular ecology (Elsevier, Oxford, 2003) pp 104-110.  Back to cited text no. 13      
14.M.J. Reigosa, N. Pedrol and L. Gonzalez, Allelopathy, a physiological process with ecological implications (Springer, Dordrecht, 2006) pp 465-537.  Back to cited text no. 14      


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2]



 

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