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DNA Profile and Bioactivities of Salsola vermiculataL. Growing in EgyptEl Zalabani S.M. 1, Koheil M.A. 2, El-Hefnawy H.M. 1,2, Farag M.A.1 & Rasheed D.M. 2

1Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Kasr El-Aini 11562, Egypt2Department of Pharmacognosy, Faculty of Pharmacy, October 6 University, Sixth of October, Central axis, Part 1/1, Egypt

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13

Photograph ofSalsola vermiculata L. (Flowering br

Introductions Salsola is represented in Egypt by 12-14 species growing wild in saline soils, the most common are S. kali L., S. tetrandraForssk. and S. vermiculataL.(1). In fcine, the plants are used for treatment of a certain skin cancerous condition, as well as cathartic, diuretic, emmenagogue, stimulant and vermifuge; meanwhile, young plae as a fodder (2). Literature reports account for the presence of flavonoids, triterpenes and alkaloids in plants of this genus (2,3) ; however, those concerning either tposition or bioactivities of S. vermiculata are scarce. The present study aimed primarily to evaluate the biological potential of the cited plant in view to isolate ctive metabolites. Genetic characterization of the plant material was first performed through DNA profiling.

Materials and Reagentsmaterial : Flowering aerial parts (AP) and roots (RT) of S. vermiculata were gathered in January (2008) from Marsa Matrouh shade-dried and pulverized.profiling: The 10 primers used for RAPD analysis (Random Amplified Polymorphic DNA), were purchased from Operon Technologies Inc., Alameda, California, USA.gical evaluation:n tumor cell lines (hepato-cellular carcinoma HEPG2, breast adenocarcinoma MCF7 and colorectal carcinoma HCT116) were obtained from the American Type Culturection and maintained at Tumor Biology Department, National Cancer Institute, Cairo, Egypt.Diphenyl-1-picrylhydrazyl (DPPH) was purchased from (Aldrich, USA); while 2-Thiobarbituric Acid, Nicotinamide Adenine Dinucleotide (reduced form, NADH), Nitro Blueazolium (NBT), Phenazine Methosulphate (PMS), Butylated Hydroxytoluene (BHT) and -Tocopherol from Sigma Chemical Company (St. Louis, Mo, USA).

Preparation and Examination of Plant Extractsdried aerial parts (AP) and roots (RT) (1 Kg, each) were extracted using 70% methanol. The methanol extract (ME) of aerial parts (AP) was fractionated between n-ne (HE), methylene chloride (MC), ethyl acetate (EtOAc) and n-butanol (Bu). The percentage yield of ME was 10% while those of the fractions were HE 0.66, MC 0.37,c 4.26 and Bu 3.33%, respectively. Phytochemical screening of the different extracts was performed using conventional methods (4).

DNA Fingerprinting

edure:Ten primers; OPA02, OPA03, OPA17, OPA20, OPE02, OPE18, OPO16, OPB06, OPG02,03 were used for RAPD analysis. DNA was extracted from fresh plant material, treated withase to remove RNA from sample, then RAPD-PCR reactions were conducted under optimizedtion conditions, followed by gel electrophoresis. A marker containing a total of 20 bands from0 to 100 bp was used (5, 6).Results are demonstrated in table(1) and fig.(1).

1KbMarke

r

OP-A02

OP-A03

OP-A17

OP-A2o

OP-E02

OP-E18

OP-O16

OP-B06

OP-G02

OP-G03

Fig. (1): The obtained RAPD-PCR products for SalsolavermiculataL., using ten primers.

No. PrimerRAPD

fragments

Monomorphic

fragments

Polymorphic

fragments

Level of

polymorphism

1. OPA-02 9 3 6 66.6 %

2. OPA-03 12 4 8 66.6 %

3. OPA-17 8 3 5 62.5 %

4. OPA-20 6 3 3 50 %

5. OPE-02 9 5 4 44.4 %

6. OPE-18 11 9 2 18.2 %

7. OPO-16 6 4 2 33.3 %

8. OPB-06 3 0 3 100 %

9. OPG-02 8 2 6 75 %

10. OPG-03 5 3 2 40 %

Total 77 36 41 53.2 %

Table (1): Total numbers of RAPD-PCR fragments and distribution ofmonomorphic (common) and polymorphic bands generated by ten

primers in Salsola vermiculataL.

sults & Discussion: DNA fingerprint revealed 9 primers to be specific markers forracter ization of the plant (OPA02,OPA03,OPA17,OPA20,OPE02,OPE18,OPO16,OPG02, OPG03)a level of polymorphism of 53.2%. These findings serve to help in the genetic characterization

the plant and the genus.

Cytotoxic ActivityProcedure: The cytotoxic activity of S. vermiculata (ME) extract and fractions thereof of (and (RT) were assayed by SBR (Sulphorhodamine-B) colorimetric assay using Doxorubicin aspositive control (7). Cytotoxicity was expressed as the percentage of viable cells relative to ceincubated in 0.1% DMSO (negative control). IC50 (Inhibitory Concentration 50): concentratsufficient to produce 50% inhibition of the cell line growth was determined. The survival fractwas calculated from the following equation:

Surviving fraction = (CI) of treated cells / (CI) of control cellswhere (CI) is the color intensity, measured spectrophotometrically by ELISA microplate reader a

564 nm. Results are illustrated in fig. (2, 3).

0

5

10

15

20

25

30

35

40

45

70%alc.ext. n-hexanefraction Chloroform

fraction

Ethylacetate

fraction

n-Butanol

fraction

Doxorubicin

standard

HEPG2

MCF7

HCT116

IC50

(g/ml)

Fig (2): Cytotoxic activity of t he total alcoholic extract and thefractions thereof of S. vermiculataL. (AP) against different cell

lines as determined by SRB assay.

0

5

10

15

20

25

30

35

40

45

70%alc.ext. n-hexane

fraction

Chloroform

fraction

Ethylacetate

fraction

n-Butanol

fraction

Doxorubicin

standard

HEPG2

MCF7

HCT116

IC50

(g/ml)

Fig (3): Cytotoxic activity of the total alcoholic extract and thefractions thereof of S. vermiculataL. (RT) against different

cell lines as determined by SRB assay.

Results & Discussion:ME extracts and fractions of S. vermiculata L. AP and RT exhibitemoderate to high cytotoxic activity against three carcinoma cell lines (MCF7, HCT116 aHEPG2) in a dose dependant range of 8.6 42.8 g/ml. AP extract showed a marked activagainst HEPG2 and MCF7, whereas RT extract showed the highest cytotoxic activity againHCT116 by Bu fraction (IC50 = 8.6 g/ml) and a moderate activity against MCF7 and HEPG2 ME and HE extracts (IC50 = 12.5 and 13.4 g/ml respectively).

Radical Scavenging Activitycedure for DPPH assay: The assay measures the degree of reduction in absorbance of theH alcoholic solution in the presence of the tested sample or D, L- -tocopherol and (BHT) controltions at 517 nm (8). Percentage inhibition of the DPPH color was calculated according to thewing equation:

% Inhibition = 1 - [Ab sample /Ab Blank) X 100]

cedure for superoxide anion (SO) scavening activity: SO radicals generated by phenazinehosulphate-nicotinamide adenine dinucleotide (PMS-NADH) systems, were determined by monitoringproduct of nitroblue tetrazolium (NBT) spectrophotometrically at 560 nm (9). The decrease in colorr addition of the tested and control samples; D, L--tocopherol and (BHT) is a measure of theirscavenging activity. Percentage inhibition was calculated according to the following equation:

% Inhibition= [(AC-At)/AC] x 100ults are illustrated in fig. (4,5).

0

10

20

30

40

50

60

70

80

90

70% Alc.

Ext.

Chloroform

fraction

Ethyl acetate

fraction

Butanol

fraction

D,L,-

tocopherol

BHT

77.21.9

70.51.8

57.21.562.21.5

64.81.5

55.51.4

%

Decoloration

0

10

20

30

40

50

60

70

80

90

70% Alc.

Ext.

Chloroform

fraction

Ethyl acetate

fraction

Butanol

fraction

D,L,-

tocopherol

BHT

79.51.9

75.21.8

65.51.548.21.3

68.51.6

52.21.3

%

InhibitioninPMS-NADHsystem

(4): Inhibition of DPPH by alcoholic extract and fractions of SalsolavermiculataL. Aerial parts.

Fig (5): Inhibition of SO anion by alcoholic extract and fractions ofSalsola vermiculataL. Aerial parts.

Results & Discussion:AP extracts had a significant scavenging effects on both, DPPH andSO anion. Percentage inhibition of DPPH and SO by ME extract and was found to be (77.2%and 79.5%) and its MC fraction exerted (70.5% and 70.5%). Both results exceeded that of -tocopherol (64.8% and 68.5%) and BHT (55.5% and 52.2%).

Anti Lipid Peroxidation ActivityProcedure: Malondialdehyde (MDA) was measured in untreated control rat liver tissue homogenas thiobarbituric reactive substance (TBARS). Induction of lipid peroxidation with FeSO4/Hcaused an elevation in the levels of TBARS. Percentage inhibition of TBARS after treatment withvermiculata AP (ME) and fractions was calculated according to the following formula (10) :

% Inhibition = 1 [MDA content in sample / MDA content in control X 100]

Results are illustrated in fig. (6)

0

10

20

30

40

50

60

70

70% Alc. Ext . Chloroform

fraction

Ethyl acetate

fraction

Butanol fraction D,L,-

tocopherol

BHT

57.11.5

65.71.6

40.31.238.31.2

48.81.3

43.11.3

%

InhibitionofMDAcontent

Fig (6): Inhibition of MDA content by Salsola vermiculataL. aerial parts extract and fractionsin rat liver tissue homogenate.

Results & Discussion:The antioxidant activity against lipid peroxidation was also assesseby montoring of the percentage inhibition in MDA content. ME extracts and fractions of Svermiculata L. AP significantly reduced MDA levels indicating anti-lipid peroxidation activitieThe inhibition percentages were in the range of (38.365.75%). Strongest inhibition wobserved by the MC fraction, exceeding that of -tocopherol and BHT (48.8% and 43.1respectively).

Conclusions study provides further evidence for the remarked cytotoxic and antioxidant activities of Salsola vermiculata L. The antioxidant activity was assessed by more than oel viz. radical scavening activity of DPPH and SO anions and inhibition of lipid peroxidation. These activities could be attributed to the phenolic constituents detected in tted extracts. Isolation of phytoconstituents mediating for the observed effects is currently under investigation.

References1. Turki,Z.A. Chemotaxonomical studies of the genus Salsola(Chenopodiaceae) in Egypt.,Egypt.J.Bot. 38 (1-2): 47-61 (1998).2. Duke, J.A.Handbook of energy crops, unpublished , (1983).3. Oueslati,-M-H; Jannet,-H-B; Mighri,-Z; Chriaa,-J and Abreu,-P-M. "Phytochemical constituents from Salsola tetrandra." J.Nat.Prod.69(9): 1366-1369, (2006).4. Trease G.E. and Evans, W.C. ; Pharmacognosy 15thedn. Saunders publishers , London 1989.

5. Junghans, S. and M. Metzlatt; "A Simple and Rapid Method For The Preparation of Total Plant DNA.", Biotechniques, 8, 176, (1990).6. Williams, JGK., Kubelik, AR., Livak, KJ., Rafalski, JA., Tingey, SV.; "Random Amplified Polymorphic DNA (RAPDs).",Nuc. Acids Res., 18, 6531-6535, (1990).7. Skehan, P. and Storeng, R.; "New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening.", J. Natl. Cancer Inst., 82, 1107-1112, (1990).8. Hosny, M. and Rosazza, J.P., "J. Agric. Food Chem.,50 , S639 S645, (2002).9. Liu, F., Ooi, V.E. and Chang, S. T. "Life Science", 60, 763-771, (1997).10. Hino, T., Kawanish, S., Yasui, H., Oka, S. and Sakvrai, H., "Biochem. Biophys. Acta.", 1425, 47-60, (1998).