Raju A et al.

, IJSID, 2012, 2 (2), 316-324

ISSN:2249-5347

IJSID

International Journal of Science Innovations and Discoveries

Research Article
1 Shri

An International peer Review Journal for Science

Available online through www.ijsidonline.info

Raju A1*, Christina AJM2

IN VITRO ANTIOXIDANT AND ANTICANCER ACTIVITY OF DROSERA BURMANNII VAHL (DROSERACEAE) Rawatpura Institute of Pharmacy, Datia, Madhya Pradesh, India; 2 AIMST University, Malayasia
ABSTRACT

Received: 16.02.2012 Accepted: 30.04.2012

*Corresponding Author

Drosera burmannii vahl was evaluated in a series of assay involving free radicals and Reducing power. Analysis of the free radical scavenging activities of the fractions

reactive oxygen species and IC50 values were determined. Both the extracts showed

scavenging effect on Hydroxyl radicals, DPPH Assay, Super oxide radical scavenging activity, Chelating ability of ferrous ion, Nitric oxide radical inhibition, ABTS and ABTS+, DPPH, NO, OH+ and SO radicals to non-radical form. Ascorbic acid acts as results obtained in the present study indicated that D. burmannii Vhal. extacts can be a potential source of natural antioxidant and anticancer drug. standard in the scavenging activity. In-vitro anticancer activity was evaluated by using revealed a concentration-dependent and antiradical activity resulting from reduction of

In vitro Antioxidant and anticancer activity of ethanol and aqueous extracts of

Address: Name: Raju A Place: Department of Pharmacology, Shri Rawatpura Institute of Pharmacy, Datia, M.P. E-mail: rajuasirvatham@yahoo.com

Trypan blue exclusion method and Lactate dehydrogenase (LDH) leakage assay .These Keywords: Antioxidant, Drosera burmannii Vhal, Radical scavenging, Trypan blue exclusion, Lactate dehydrogenase (LDH) leakage assay.

INTRODUCTION

INTRODUCTION

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Raju A et al., IJSID, 2012, 2 (2), 316-324 proteins, lipids, lipoproteins and DNA1.Under normal circumstances, the reactions of ROS are detoxified and controlled by a ROS are produced and despoiled by all aerobic organisms. ROS can readily react with most biomolecules including INTRODUCTION

system of enzyme such as superoxide dismutase, catalase and glutathione peroxidases 2.Oxidative stress (OS) results from an imbalance between the generation of reactive oxygen species and endogenous antioxidant systems. When the mechanism of increase the antioxidant capacity of the plasma and reduce the risk of certain diseases such as cancer, heart diseases and stroke5. The secondary metabolites like phenolics and flavonoids from plants have been reported to be potent free radical scavengers. antioxidant protection becomes unbalanced by exogenous and endogenous factors, it results in inflammation, diabetes,

genotoxicity, cancer and accelerating aging3. Medicinal plants are an important source of antioxidants 4. Natural antioxidants Here one of such a plant is Drosera burmannii vahl. Genus Drosera (Droseraceae) commonly called sundew,consists

of approximately 170 species6. Three species of Drosera Vahl are found in India viz., Drosera burmannii Vahl, Drosera indica L., flavonoides, which have pharmacological value. Plumbagin is used for curing bronchialinfection, whooping cough,

and Drosera peltata J.E.Sm7, 8. The genus Drosera contains naphthoquinones such as plumbagin,7-methyljuglone and hyperglyceamia, hypolipidaemia, uberculosis, spasms, microbial infections,leprosy, leishmaniasis, malaria, cancer, fertility insecticide, antifeedant, abortifacient and enhancesin vitro phagocytosis of human granulocytes and inhibits the development of insect and parasitic nematodes. 7- methyljuglone is shown to be inhibitory to several insects and highly toxic to fungal vahl (AEDB). antioxidant and anti-cancer potential of Ethanol Extraxt of D. burmannii vahl (EEDB) and Aqueous Extract of D. burmannii MATERIAL AND METHODS Plant material

problems, arteriosclerosis, phthisis, asthma, and acts as immunomodulator, cosmetic, aphrodisiac, chitin synthetase inhibitor, pathogens. Quercetin, one of the flavonoids, is active against cancer 9. The aim of the present work was to evaluate in vitro

material was identified and authenticated by Dr. S.N.Yoganarasimhan, Taxonomist and Research Coordinator at M. S. Ramaiah College of Pharmacy, Bangalore, Karnataka, India. The material was washed, shade dried, powdered, passed through sieve no. 60 and stored in air tight containers for experimental work. Alcoholic extract extractor. The extract(20.2g) was concentrated in a rotary flash evaporator at a temperature not exceeding 50C. Aqueous extract concentrated in a rotary flash evaporator at a temperature not exceeding 60C. In vitro Antioxidant methods 1. DPPH (1, 1 diphenyl 2, picryl hydrazyl) Method10,11 A weighed quantity (300 g) of the air-dried powdered drug was taken and extracted with ethanol (90 %) in a Soxhlet A weighed quantity (300 g) of the air-dried powdered drug was taken, macerated with hot water at 80c. The

The whole plant of D. burmannii Vahl was collected from the forests of Savanadurga, Karnataka, India. The plant

maceration process was carried out for 24 h. The macerate was filtered through Whatman No.1 filter paper. The filtrate was

concentrations of the extracts were added in a mixture contain 1ml of (0.1mM) DPPH in ethanol, 550 l of 50 mM Tris HCl International Journal of Science Innovations and Discoveries, Volume 2, Issue 2, March-April 2012

This is the most widely reported method for screening of antioxidant activity of many plant drugs. Different

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buffer pH (7.4) and was incubated for 30 min at room temperature. The procedure involves measurement of decrease in as absolute control 2. Super Oxide radical scavenging activity 12,13

Raju A et al., IJSID, 2012, 2 (2), 316-324

absorbance of DPPH at its absorption maxima of 516 nm, which is proportional to concentration of free radical scavenger added to DPPH reagent solution. The activity is expressed as inhibitory concentration (IC 50). Mixture without substrate served ml of NADH solution ( 468 M of NADH dissolved in 1 ml of 100 mM phosphate buffer pH 7.4) and 0.1 ml of various started by adding 100 l of Phenazine methosulphate (PMS) solution (60 M of PMS in 100 l of 100 mM phosphate buffer , pH 7.4). The reaction mixture was incubated at 25C for 5 min and the absorbance was measured at 560 nm. 3. Hydroxyl radical scavenging activity 14,15 Hydroxyl radical scavenging capacity of an extract is directly related to its antioxidant activity. This method involves 1ml of Nitroblue tetrazolium (NBT) solution (156 M NBT dissolved in 1.0 ml of 100 mM phosphate buffer , pH 7.4), 1

concentration of extracts and the standard Ascorbic acid were thoroughly mixed in different test tubes and the reaction

in-vitro generation of hydroxyl radicals using Fe3+/ascorbate/EDTA/H 2O2 system using Fenton reaction. In one of the methods the hydroxyl radicals formed by the oxidation is made to react with DMSO (dimethyl sulphoxide) to yield spectrophotometrically against reagent blank. 4. Nitric oxide radical inhibition activity 16 formaldehyde. Formaldehyde formed produces intense yellow color with Nash reagent (2M ammonium acetate with 0.05M acetic acid and 0.02M acetyl acetone in distilled water).The intensity of yellow color formed is measured at 412nm

concentrations of the plant extract were incubated at 25C for 150 min. At the end of incubation, 0.5ml of the reaction mixture was removed and 0.5 ml of Griess reagent (1 % sulfanilamide, 2 % H3PO4, and 0.1 % naphthylethylene diamine dihydrochloride) was added. The absorbance of chromophore formed was measured at 546 nm. 5. Reducing Power Method 17 potassium ferricyanide [K Fe (CN)] (10g/l), then mixture was incubated at 50 0 C for 20 minutes. 2.5 ml of trichloroacetic acid solution was mixed with 2.5 ml of distilled water and 0.5 ml FeCl 3 (1g/l) and absorbance measured at 700 nm. Increased absorbance of the reaction mixture indicates stronger reducing power. 6. ABTS (2, 2-azinobis (3-ethyl benzothiazoline-6-sulfonicacid) diamonium salt) Method. The ABTS assay is based on inhibition of the absorbance of radical cation, ABTS+ at 734 nm, by antioxidants. In this Different concentrations of plant extracts solution was mixed with 2.5 ml phosphate buffer (0.2 M, pH 6.6) and 2.5 ml

Reaction mixture (3 ml) containing sodium nitroprusside (10 mM) in phosphate-buffered saline (PBS) and various

(100g/l) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. Finally, 2.5 ml of the supernatant

assay, the ABTS cation radical was generated using potassium persulphate. The absorbance of the formed chromophore was measured at 734 nm. The colored radical when mixed with antioxidant, is converted to the colorless ABTS. The percentage inhibition was calculated after measuring the absorbance at 734 nm 18,19. 7. Metal(Fe2+) chelating assay 20 the absorbance of the solution at 562 nm. Plant extracts, 5 ml was added to a solution of 0.1 ml of 2 mM FeCl 2. This was followed by the addition of 0.2 ml of 5

mM ferrozine solution, which was left to react at room temperature for 10 min under shaking conditions before determining International Journal of Science Innovations and Discoveries, Volume 2, Issue 2, March-April 2012

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Raju A et al., IJSID, 2012, 2 (2), 316-324 In Vitro Anticancer Activity assay21, 22. Short term cytotoxicity was assessed by Trypan blue exclusion method and Lactate dehydrogenase (LDH) leakage

Trypan blue exclusion method

burmannii vahl. EEDB and AEDB were dissolved in distilled water. Different concentration (200, 100, 50, 25 and 10mcg/ml)) finally 100l (1X106 in 1 ml) of both the Daltons Ascitic Lymphoma (DAL) and Ehrlich Ascitic Carcinoma (EAC) was added. 30minutes. About 100l of tryphan blue dye was added to all the test tubes. Then the number of dead cells was counted by using a haemocytometer under a compound microscope. Percentage of cytotoxicity was calculated by the following formulae Lactate Dehydrogenase (LDH) leakage assay % dead cells = Number of dead cells/sum of dead cells and living cell 100.

of both extracts were prepared. In a test tube, 100l of plant extracts were added with 800l of phosphate buffer saline and

Tryphan blue dye assay method 22,23 was carried out to know the in vitro cytotoxicity potential of both extracts of D.

Each concentration of extracts did in a triplet manner. Then all the samples were incubated at 370C in an incubator for

protocol in the users manual. To determine IC50, different concentrations of herbal extracts as well as positive control 5-FU as

stated above were incubated with 100 l of DLA and EAC cell suspension having 1x10 6 cell/ml in 96 well plates and incubated is the absorbance of the control sample. Statistical analysis

at 37C for 4 hrs in 5% CO2 atmosphere. All the control and test substances were tested in triplicates and mean SEM of the absorbance value were taken to calculate cytotoxicity. LDH leakage (%) related to control wells containing cell culture medium For all the experiments three samples were analyzed and all the assays were carried out in triplicate. The results were Where AC Absorbance of Control; AT- Absorbance of Test RESULTS AND DISCUSSION Table 1. In vitro Antioxidant effect of EEDB and AEDB Extract Concentration Used (mcg) IC50 Concentration of EEDB (mcg) 27.20.64 57.60.97 144.92.41 52.80.92 64.780.45 64.10.23 15.21.54 % Inhibition = [(AC-AT) / AC 100]

LDH leakage assay was carried out using LDH cytotoxicity detection kit by Sigma Aldrich Inc., USA, according to

without extracts was calculated by [A]test/ [A]control X100. Where [A]test is the absorbance of the test sample and [A]control expressed as mean standard deviation. % Inhibition of all the methods were calculated from their respective absorbance to the corresponding concentration of extracts by following formula

The results of in vitro anti-oxidant showed in Table no 1. The EEDB and AEDB have shown remarkable antioxidant activity. S no. 3 4 5 6 7. 1 2 DPPH Assay Hydroxyl Scavenging Activity NO Scavenging Assay Fe2+ Chelating Assay Reducing Power Assay Super Oxide Radical Assay Radical Scavenging of ABTS In vitro Model IC50 Concentration of AEDB (mcg) 88.82.88 64.21.78 320.410.78 641.5 448.52.64 124.70.78 76.80.35 IC50 Concentration of AA (mcg) 85.710.82 110.031.02 95.560.78 160.780.26 110.120.18 76.80.35 1701.42

5,10,20,40,80,160,320, 640

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Raju A et al., IJSID, 2012, 2 (2), 316-324

100

80

DLA EEDB EAC EEDB EAC AEDB DLA AEDB

cell count (dead)

60

40

20

0
50 10 5 Co nt ro l
DAL AEBD DALEEBD EAC EEBD EAC AEBD
C O N 5

25 0

15 0
100 80 60 40 20 0

Concentration (mcg/ml)

Graph 1 : Trypan blue exclusion method

% cytotoxicity

10 0

50

25 0

15 0

Concentration (mcg/ml)

Graph 2: LDH leakage assay

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Raju A et al., IJSID, 2012, 2 (2), 316-324 In vitro antioxidant Assay active oxygen species. In this study certain non-enzymatic assays reduced the free radical formation and IC 50 values were shown in Table 1. IC50 values were calculated by interpolating the extracts concentration vs % inhibition. It is defined as the concentration of extracts necessary to decrease the absorbance of chemicals by 50%. DPPH is relatively stable nitrogen centered free radical that can accept an electron or hydrogen radical to become a stable diamagnetic molecule. DPPH radicals react with suitable reducing agent as a result of which electron become paired off electrons consumed. EEDB (64.10.23) showed high DPPH radical scavenging activity than AEDB (124.70.78) and standard antioxidant ascorbic acid showed 1701.42 of inhibition. systems, super oxide anions derived from dissolved oxygen by PMS/NADH coupling reaction reduce NBT
25, 26.

Antioxidant systems are very important for shielding cellular membranes and organelles from the damaging effects of

forming the corresponding hydrazine24. The solution therefore loses color; intensity of color is depending on the number of

DPPH assay is based on the measurement of the scavenging ability of antioxidant towards the stable DPPH radical.

NBT is the most popular method. The method is based on generation of super oxide radical which reduces NBT to a blue colored formazon that can be measured at 560nm The decrease in the absorbance with the antioxidants indicates the radical scavenging activity than AEDB (641.5) and standard antioxidant ascorbic acid showed 160.780.26. saline and measured by Griess reagent
27.

The formation of super oxide radical leads to a cascade formation of other ROS in the cell. In the PMS/NADH-NBT Reduction of

consumption of super oxide anions in the reaction mixture. In the presence study EEDB (52.80.92) showed high super oxide 546nm. This scavenging power is may be due to the anti-oxidative principles in the extracts which compete with oxygen to react with nitric oxide thereby inhibiting the generation of nitrite. Our experiments showed EEDB (27.20.64) showed high NO scavenging radical scavenging activity than AEDB (88.82.88) and standard antioxidant ascorbic acid showed 85.710.82. were found to be lesser than AEDB (320.410.78), indicating that it EEDB has more reducing power than AEDB. acid and ferric chloride, which is measured at 700nm, where in presence of reductants (antioxidants) in the plant extracts, causes the reduction of Fe3+/Ferricyanide complex to ferrous form. The mean values of EEDB (144.92.41) concentration In reducing power assay antioxidant compound forms a colored complex with potassium ferricyanide, trichloro acetic In presence of scavengers, the absorbance of the chromophore is evaluated at

NO scavenging assay is based on the inhibition of nitric oxide radical generated from sodium nitroprusside in buffer

In Metal chelating assay Ferrozine can quantitatively form complexes with Fe2+. The absorbance of Ferrozine-Fe2+ complex decreased linearly in a dose-dependent manner. Chelating agents that forms bonds with a metal are effective as secondary work, EEDB was found to reduce inhibitory concentration (57.60.97) as compared to AEDB (64.21.78), whereas standard ascorbic acid showed at a concentration of 110.031.02. 76.80.35. antioxidants because they reduce the redox potential, and thereby stabilize the oxidized form of the metal ion. In the present yielding malondialdehyde, which is mutagenic and carcinogenic. In the present work, EEDB was found to reduce inhibitory Hydroxyl radical is the most reactive radical in the ROS system. It initiates the peroxidation of cell membrane lipids The principle behind the ABTS assay technique involves the reaction between ABTS and potassium persulphate to International Journal of Science Innovations and Discoveries, Volume 2, Issue 2, March-April 2012

concentration (15.21.54) as compared to AEDB (76.80.35), whereas standard ascorbic acid showed at a concentration of converted back to colourless ABTS, the absorbance of which is measured at 734nm. The free radical scavenging activity by this

produce the ABTS radical cation (ABTS+) a blue green chromogen. In the presence of extracts, the coloured radical is

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method of EEDB (64.780.45) showed significant inhibitory concentration when compared with AEDB (448.52.64) and Ascorbic acid (110.120.18). respectively. In-vitro anticancer activity Ascitic Carcinoma (EAC) were shown in Figure no 1 and 2 as trypan blue exclusion method and LDH leakage assay method was compared with control. LDH leakage assay showed the % cytotoxicity of EEDB and AEDB against the cell lines DAL and showed 58.03% on DLA and 57.6% on EAC cell line. In trypan blue exclusion method, 250, 150,100 mcg/ml of EEDB and AEDB were more significant (p<0.001) against Anticancer activity of EEDB and AEDB against the test cells namely Daltons Ascitic Lymphoma (DAL) and Ehrlich

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DAL, whereas 50,10 and 5 mcg/ml EEDB and AEDB showed p<0.01 significant on DAL and EAC. The inhibition concentration In the present study % of LDH release increased with increase in concentration of both extract. Lactate

EAC. At 250mcg, concentration of ethanol extract has showed 67% on DLA and 93% on EAC cell line, whereas aqueous extract dehydrogenase is a glycoprotein enzyme and is release into the medium when cells die and lost their membrane integrity. The release of LDH in the medium and change (decrease) of activity over time indicates cells dead and loss of membrane integrity,

and a value of 100% LDH release is an indication that the type of cell death induced is necrotic cell death. This is because the activity at minimum concentration of both the extracts. But ethanol extract showed a remarkable increase in percentage of dead cells than the aqueous extract. CONCLUSION activity. The overall antioxidant activity of these extracts might be attributed to its phytochemical constituents. These could be a source of natural antioxidant that could have greater importance as therapeutic agent in preventing or slowing oxidative stress related degenerative diseases. The plant also showed anticancer activity against cell lines. 1. 2. 3. 4. 5. 6. 7. REFERENCES Gilg. African Journal of Pharmacy and Pharmacology 2010; 4(2): 70-78. Res. 2001; 44: 491-495. 2004; 36: 827 828. 35: 588 592. Oyedemi SO, Bradley G, Afolayan A J. In -vitro and in-vivo antioxidant activities of aqueous extract of Strychnos henningsii Arouma OI. Characterization of drugs as antioxidant prophylactics. Free Radical Biol and Med 1996; 20(5): 675-705. Buyukokurogla ME, Gulein L, Oktav M, Kufrevioglu OI. In vitro antioxidant properties of dantrolene sodium. Pharmacol C. Rice-Evans, Flavonoids and isoflavones: absorption, metabolism and bioactivity, Free Radical Biology and Medicine, http://thecarnivorousplantscociety.org

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