in vitro antioxidant and free radical scavenging activities.pdf

Journal of Pharmacy Research Vol.5 Issue 6.June 2012

B.R. Srilatha et al. / Journal of Pharmacy Research 2012,5(6),3296-3303

3296-3303

Research ArticleISSN: 0974-6943

Available online throughwww.jpronline.info

*Corresponding author. B.R.SrilathaDepartment of Studies in Chemistry,University of Mysore,Manasagangotri,Mysore-570006,Karnataka, India

In Vitro Antioxidant and Free Radical Scavenging Activitiesof Mukia Maderaspatana (Linn.) M.Roem

B.R. Srilatha* and S. AnandaDepartment of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore-570006, Karnataka, India

Received on:14-02-2012; Revised on: 22-03-2012; Accepted on:08-04-2012

ABSTRACTMukia maderaspatana (Linn.) M. Roem, is an annual monoecious, climbing vine or prostrate herb, and is an edible plant. It is also extensively used inFolklore medicine. In this study the antioxidant and free radical scavenging activities of the plant extract are characterized. Methanol extract of the edible partof the whole plant was prepared and studied for its total phenolics and flavonoid content, invitro antioxidant activities lipoxygenase and cyclooxygenseinhibiting activities and free radical scavenging activities. Hydroxyl radical, nitric oxide, superoxide and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radicalscavenging activities were determined by standard methods and compared with appropriate reference compounds like ascorbic acid, catechin or butylatedhydroxyl anisole (BHA).The antioxidant activities of M. maderaspatana were observed in a dose-dependent manner. The total antioxidant and reducingpower activities were comparable to those of ascorbic acid and BHA. Cyclooxygenase (COX), lipoxygenase (LOX) inhibiting activity and the hydroxylradical, nitric oxide, superoxide and DPPH radical scavenging activities were also comparable to the reference compounds like ascorbic acid, catechin,quercetin and BHA. The cyclooxygenase activity and lipoxygenase activity of macrophages and the lipoxygenase activity of soybean extract were alsoinhibited. The plant extract had phenolics and flavonoids.M. maderaspatana has a powerful antioxidant activity against various in vitro oxidative systemsand would probably be equally effective against in vivo radicals and oxidants. Since M. maderaspatana is an edible plant, it can be a source of naturalantioxidants and be useful as potential food supplement.

Keywords: Mukia maderaspatana, antioxidant activity, LOX, COX, free radical scavenging.

INTRODUCTIONIn healthy individuals, the production of free radicals is balanced by theantioxidative defence system. Free radical generation is one of the antibacte-rial defences and hence a natural process of the host cells. When the equilib-rium favours the generation of free radicals, it results in depletion of antioxi-dants and results in oxidative stress. Oxidative stress contributes to diversedisorders including diabetes, cardiovascular diseases, cancer and injury to thecentral nervous system.[1] Free radicals especially oxygen derived radicals areformed by one electron reduction of molecular oxygen and are collectivelycalled as Reactive Oxygen Species (ROS). ROS cause membrane lipidperoxidation.[2] In addition they can cause the oxidation of protein, DNA andother biological molecules.[3]

The lipoxygenase (LOX) enzymes catalyze the oxidation of polyunsatu-rated fatty-acids such as linoleic acid or arachidonic acid (AA) containing thecis-methylene interrupted diene structure yielding conjugated hydroperox-ides are non-heme, non-sulfur iron dioxygenases.[4] They are widely distrib-uted throughout plants, animals, fungi and some bacteria. This family ofenzymes plays a major role in polyunsaturated fatty acid metabolism bycatalyzing the incorporation of molecular oxygen into certain polyunsatu-rated fatty acids, producing hydroperoxide products. These lipoxygenasesare mediators of inflammation along with another oxygenase theCyclooxygenase (COX). Cyclooxygenase (also called Prostaglandin H Syn-thase or PGHS) is a bifunctional enzyme exhibiting both COX and peroxi-dase activities. The COX component converts arachidonic acid to a

hydroperoxy endoperoxide (PGG2), and the peroxidase component reducesthe endoperoxide to the corresponding alcohol (PGH2), the precursor ofProstaglandins, thromboxanes, and prostacyclins.[5]

In an attempt to overcome ROS induced cellular damage antioxidant supple-ments and food rich in antioxidants are included in the diet. Synthetic antioxi-dants are suspected to have side effects like liver damage and carcinogenesisin laboratory animals.[6] Hence, natural antioxidants from plant extracts likegreen tea, spice extracts and herbal extracts are studied extensively for nontoxic molecules. Plants and plant extracts have been extensively used becauseof their medicinal properties to prevent and cure diseases. 80 % of the worldspopulation still depends on natural products for treatment. Indian medicinalsystem also depends on the use of plants. Phytochemicals present in plantshave been shown to have diverse biological activities like cardioprotective,[7]

cancer prevention[8] and inhibiton of bone resorption.[9] One of the mostcommon activities of the phytochemicals is the antioxidant and free radicalscavenging activity.

Mukia maaderaspatana (L.) M. Roem (Cucurbitaceae) is an annualmonoecious, climbing vine or prostrate herb, densely covered with whitehairs. M. maderaspatana is traditionally used as a leafy vegetable and it isextensively used in Folklore medicine as good diuretic, stomachic (a digestivetonic), gentle aperient, antipyretic, antiflatulent, antiasthmatic andantibronchitis. In scientific literature M. maderaspatana has been shown tobe hepatoprotective, [10] anti-inflammatory, [11] antiarthiritic, [ 1 2 ]Immunomodulatory,[13] anti platelet [14] and antimicrobial.[15]The consumptionof M. maderaspatana leaf tea decreased the blood pressure and showedbeneficial effects on lipid profile, fibrinogen, bilirubin and body mass index inhuman volunteers. [16] This plant is edible, and is grown in the wild as well asin the kitchen garden, and since it has many health promoting activities, thepresent study was carried out to evaluate the antioxidant and radical scavengingactivities of methanolic extract of the edible part of the plant.



3296-3303

(0.1ml) was added to 1ml of methanol followed by 5 ml of chromogen reagent(1g 4 dimethylaminocinnamaldehyde dissolved in a cooled mixture of 250mlof concentrated HCL and 750 ml of methanol, completed to 1L with metha-nol). After 10 min incubation at RT, the absorbance of the reaction mixturewas measured at 640nm. Catechin was used as a standard. Total flavonoidcontent was expressed as mg catechin equivalent per gm of extract.

IN VITRO ANTIOXIDANT ASSAY

Total antioxidant activityThe total antioxidant activity of sample was evaluated by the method of

Estimation of total flavonoids contentThe total flavonoids were estimated using quercetin, phloroglucinol or cat-echin as standards by three different methods as follows:Aluminium chloride (ALCL3) reagent method of Chang et al

[19]: The plantextract (0.1ml) was mixed with 1.5ml of methanol, 0.1ml of 10% aluminiumchloride, 0.1 ml of 1M potassium acetate and 2.8ml of distilled water. After30 min incubation at RT, the absorbance of the reaction mixture was mea-sured at 415nm. Quercetin was used as standard. The flavonoid content wasexpressed as mg quercetin equivalent per gm of extract.

Vanillin reagent method of Swan-Hillis [20]. The plant extract (0.1ml) wasadded to 1 ml of 50% methanol followed by 1.5 ml of vanillin reagent .Afterincubation for 15min at RT, the absorbance of the reaction mixture wasmeasured at 500nm.Phloroglucinol was used as standard. The flavonoid con-tent was expressed as mg phloroglucinol equivalent per gm of extract.

Chromogen reagent method of Delcour and de Varebeke [21]. The plant extract

Preparation of Methanolic ExtractThe edible part of the whole plant was shade dried and pulverized using a

Estimation of total phenolic contentThe total phenolic content was estimated by using Folin-Ciocalteu reagentaccording to the method of Singleton and Rossi with the slightmodification.[18]Briefly,0.1ml of the plant extract was mixed with 1ml of FCreagent(1:1 with water) and incubated at RT for 5 min.1ml of 20%Na2CO3solution was added to the reaction mixture which was incubated atRT for 30 min, and the absorbance was read at 750nm.Gallic acid was used asstandard and the total phenolic content was expressed as gallic acid equiva-lents in milligrams per gram of extract.

Preliminary Phytochemical AnalysisThe extracted plant material was subjected to qualitative phytochemicalscreening. The presence or absence of the phytochemical constituents ofextracted plant material was analyzed by using the standard methods.[17]

mechanical grinder. The powdered plant material (50g) was extracted withmethanol (200ml) by soxhlet apparatus for 24 hours. The extract was evapo-rated using a rotary vaccum-evaporator at 40oC to provide dry extract, witha yield of 10% w/w. The extract was kept at -200C until use.

Plant materialM. maderaspatana were collected from Hassan district, Karnataka state,India during the month of June, authenticated by botanist Dr. M.S. Sudarshana,University of Mysore. A voucher specimen (BOT-003-2010) deposited inthe Botany Herbarium, University of Mysore. Mysore.

MATERIALS AND METHODS

ChemicalsQuercetin, catechin, Linoleic acid, Adenosine-5-triphosphate (ATP),

ene diamine tetra acetic acid(EDTA), nicotinamide adenine dinucleotide(NADH), nitroblue tetrazolium (NBT), phenazonium methosulphate (PMS)and trichloroacetic acid(TCA) were purchased from SRL(Mumbai, India).All other chemicals and solvents used are of analytical grade.

acid (TBA), 4- dimethylaminocinnamaldehyde were purchased from MERCK(Mumbai, India). Folin-Ciocalteuss phenol reagent, 2-deoxy-2-ribose, phlo-roglucinol, ascorbic acid, gallic acid (GA),vanillin, ammonium molybdate,Sodium nitroprusside , a-napthyl-ethylenediamine dihydrochloride, ethyl

Dithiothreitol (DTT), Amplex Red (N-acetyl-3,7-dihydroxyphenoxazine),1, 1-Diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma Chemi-cal Co. (Bangalore, India). Butylated hydroxyl anisole (BHA), thiobarbituiric

Cyclooxygenase inhibiting activityCycloxygenase enzyme activity was assayed using rat peritoneal macroph-ages. The rat peritoneal macrophages were isolated essentially by the methoddescribed by Suzuki and Murachi.[24] The macrophages were lysed and thelysate was used as an enzyme source. Cycloxygenase was assayed usingAmplex Red reagent by the method of Zhou et al.[25] In the presence ofcycloxygenase the non fluorescent reagent is converted to resorufin, a fluo-rescent molecule. The assay reaction was carried out in final volume of 1mlcontaining 100 M linoleic acid and 10 M Amplex red reagent(prepared inpure DMSO and stored at -20oC) in 50mM Tris HCl buffer pH 9.0. Thereaction mixture was incubated for 5 min at 37oC and the relative fluores-cence intensity was measured in a fluorimeter using appropriate blanks. Theexcitation was at 563nm and emission was at 587nm. The cycloxygenaseinhibitor assay was carried out by pre incubating the enzyme with the plantextract for 15 min prior to determining its COX activity. The results areexpressed as percent inhibition of the COX activity. The results were com-pared with standard phytochemicals.

Lipoxygenase inhibiting activityLipoxygenase enzyme activities were assayed using rat peritoneal macroph-ages as source of enzyme for 5- Lipoxygenase and soybean extract as asource for 12- Lipoxygenase.

5-Lipoxygenase inhibition:Rat peritoneal macrophages were isolated as described above. Macrophagelysate was used as a source of the 5- Lipoxygenase. The enzyme assay wascarried out essentially by the method of Aharony and Stein.[26] Briefly, thereaction was carried out in a final volume of 2ml containing 50 M DTT, 200M ATP, 300 M CaCl2, and 150 M Linoleic acid. The reaction was started

Reducing power assayThe reductive potential of the extract was determined according to the methodof Oyaizu.[23] Different concentrations of plant extract in 0.5 ml of MeOHwere mixed with 2.5 ml of Phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of1% potassium ferricyanide. The mixture was incubated for 20 minutes at 50C. At the end of the incubation, 2.5 ml of 10% trichloroacetic acid was addedto the mixture and centrifuged at 3000 rpm for 10 minutes. The upper 2.5 mllayer was mixed with 2.5ml of distilled water and 0.5 ml of 0.1% ferricchloride, and the absorbance was measured at 700 nm. A higher absorbance ofthe reaction mixture indicated greater reducing power. BHA was used as apositive control.

with 1ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phos-phate and 4 mM ammoniummolybdate). The reaction mixture was incubatedin a water bath at 95C for 90 min. After cooling to room temperature, theabsorbance of the mixture was measured at 695 nm against blank. The rela-tive activity of the sample was compared with that of standard ascorbic acid.

Prieto et al.,[22] based on the reduction of Mo (VI) to Mo (V) by the antioxi-dant compounds and formation of green phosphomolybdenum complex atacidic pH.Various concentrations (20-100g/ml) of plant extract were mixed



3296-3303

100 x A

AA

0

10

-% inhibition [OH] = (Equation 1)

Table 1: Qualitative phytochemical screening of Mukia maderaspatana extract

Plant Constituents Tested Result

Alkaloids +Flavonoids +Tannins +Saponins -Glycosides +Steroids +Phenolics +Aminoacids +Quinones +

Phytochemicals were qualitatively detected by the standardmethods.+ Present; - Absent

by adding 50l of the enzyme preparation. The formation of 5-HETE wasfollowed for 3min at 234nm at room temperature. The 5-LOX inhibitingactivity of the plant extract was determined by pre incubating the enzymewith the plant extract prior to determining its 5-LOX activity. The results areexpressed as percent inhibition of the 5-LOX activity. The referencephytochemicals were also used for their inhibitory activity on 5-LOX.

Preparation of Soybean Lipoxygenase:The soybean lipoxygenase was partially purified by the method of Axelrodet al.[27] Briefly soybean seeds were obtained from National Seed CollectionCentre and powdered with a pestle and mortar. The powder was suspendedin petroleum ether (60-80) in cold and extracted three times. The defattedpowder was dried in air and suspended in 0.2M sodium acetate buffer pH 4.5at 10% concentration (w/v) and stirred at 4oC for 1 hr. The suspension wasallowed to settle and then decanted. It was then centrifuged in a refrigeratedcentrifuge at 4oC for 10 min at 5000rpm. The clear supernatant was used asthe source of enzyme.

12- Lipoxygenase inhibition:Soybean lipoxygenase activity was assayed by the procedure of Axelrod etal,.[27].Briefly the reaction was carried out in a final volume of 3 ml containing2.9 ml of 0.1M borate buffer pH 9.0 and 50 l of 10mM Linoleic acid. Thereaction was started by the addition of 50 l of the soybean enzyme extract.The enzyme activity was measured by following the formation of the prod-uct, 12-HETE at 234nm for up to 1min. The enzyme inhibition was deter-mined by pre incubating the enzyme with the plant extract or standardphytochemicals prior to determining its 12-LOX activity. The results areexpressed as percent inhibition of the 12-LOX activity.

Hydroxyl radical scavenging assayThe hydroxyl radical (OH) scavenging activity was determined by the methodof Halliwell et al,.[28] In this assay, OH is produced by reduction of H2O2 bythe transition metal (iron) in the presence of ascorbic acid. The generation ofOH is detected by its ability to degrade deoxyribose to form products, whichon heating with thiobarbituric acid (TBA) forms a pink coloured chromogen.Addition of different concentrations of plant extract competes with deoxyri-bose for OH and diminishes the colour formation. The reaction mixture ina final volume of 2 ml, contained 0.1 ml of EDTA (1mM),0.01 ml ofFeCl3(10mM), 0,1 ml of H2O2(10mM),0.36 ml of deoxyribose (10mM),1 mlof plant extract (concentrations from 20-200g/ml), 0.33 ml of phosphatebuffer(50 mM, pH 7.4) and 0.1 ml of ascorbic acid(1mM) added in sequence.The mixture was incubated at 37oC for 1 hour. 1 ml of the incubated mixturewas mixed with 1 ml of 10% trichloroacetic acid and 1 ml of TBA (1% in0.025 M NaOH) and heated for one hour on water bath at 80oC when a pinkchromogen developed, which was measured at 532 nm. The scavenging activ-ity was compared using BHA as a standard. Decreased absorbance of thereaction mixture indicated increased hydroxyl radical scavenging activity.The percentage radical scavenging activity was calculated by using the for-mula:

it is reduced. A change in colour, from deep violet to light yellow, wasmeasured. DPPH is a stable free radical and accepts an electron or hydrogenradical to become a stable diamagnetic molecule. The intensity of the yellowcolour depends on the amount and nature of radical scavenger present in thesample and standard compounds. The reaction mixture containing 0.5 ml of0.5 mM DPPH, and various concentrations of plant extract (10- 80g/ml),were made up to 2 ml with methanol. Then the tubes were incubated at 37oCfor 30 minutes and the absorbance of this solution was measured at 517 nm.BHA was used as the standard for comparison. The percentage radical scav-enging activity was calculated by using equation 1 as described above.

Nitric oxide radical scavenging assayNitric oxide radical (NO) generated from sodium nitroprusside in aqueoussolution at physiological pH reacts with oxygen to produce nitrite ion, whichis measured by the Griess- Illosvoy reaction.[30] Briefly, 3ml of the mixturecontaining 10mM sodium nitroprusside and different concentrations of plantextract (100-600g/ml) in phosphate buffer (pH 7.4) were incubated at 25oCfor 60 min. After incubation, 0.5 ml of the reaction mixture was mixed with0.5 ml of sulfanilic acid reagent (0.33% in 20 % glacial acetic acid) andallowed to stand for 5 min for complete diazotization. Then 1ml of naphthylethylene diamine dihydrochloride (0.1% in water) was added and the solutionmixed and allowed to stand for 30 min at 25oC. A pink coloured chromophorewas formed in diffused light. The absorbance was measured at 540nm.Ascorbicacid was used as a standard. The percentage radical scavenging activity wascalculated by using equation 1 as described above.

Superoxide anion radical scavenging assaySuperoxide anion radical (O2

-) scavenging activity was determined byNishimikis method[31] with slight modifications. The assay was based on theoxidation of NADH by phenazine methosulphate (PMS) in order to liberatePMSred. PMSred converts oxidized nitroblue tetrazolium (NBToxi) to the re-duced form NBT red, which forms a violet coloured complex. The formation ofviolet colour indicated the generation of superoxide anions. A decrease in theformation of colour in the presence of the antioxidant was a measure of itssuperoxide scavenging activity. 1 ml of NBT (156 M NBT in 100 mMphosphate buffer, pH 7.4), 1 ml of NADH (468 M in 100 mM phosphatebuffer, pH 7.4), and various concentrations of plant extract (240-800g/ml),were mixed well in a final volume of 3 ml. The reaction was started by theaddition of 100 l of PMS (60 M PMS in 100 mM phosphate buffer, pH7.4). The reaction mixture was incubated at 25C for 5 minutes, and theabsorbance was measured at 560 nm. Catechin was used as the standard.Decreased absorbance of the reaction mixture indicated increased superoxideanion scavenging activity. The percentage radical scavenging activity wascalculated by using equation 1 as described above.

RESULTS

Preliminary Phytochemical AnalysisThe phytochemical analysis of the total methanolic extract revealed thepresence of various bioactive components, of which flavonoids and pheno-lics were most prominent, while it did not contain any saponins.The result ofthe phytochemical test has been summarized in Table 1.

Where A0 was the absorbance of the control and A 1 was the absorbance in thepresence of the samples or standard.

DPPH radical scavenging assayThe DPPHradical scavenging activity of the plant extract was determinedspectrophotometrically by the method of Williams et al.[29] DPPH reactswith an antioxidant compound that can donate hydrogen radical and thereby



3296-3303

Phytochemicala Standard Total content(mg equivalent/gm)b

Phenolics Gallic acid 41Flavonoids Quercetin 8.8

Catechin 0.1 Phloroglucinol 3

Table 2: Total phenolic and flavonoid contents of Mukia maderaspatana extract

a Phenolics were assayed by the method of Singleton and Rossi using Gallic acidas the standard. Flavonoids were assayed using three methods and threedifferent standards as described in methods. b Results are expressed as milligramequivalent per gram of extract.

Total phenolic and flavanoid contentTotal phenolics and flavanoids content in the methanolic extract are shown inTable 2.The phenolic content was very high compared with flavanoid con-tent. Among the flavanoids, the three different assay methods with threedifferent standards, namely Quercetin, Phloroglucinol and Catechin yieldeddifferent quantities of the flavanoids. The highest amount was detected bythe Aluminium chloride reagent using quercetin as standard and the leastamount was detected by the dimethyl amino cinnamaldehyde reagent usingcatechin as standard.

Total antioxidant capacityThe total antioxidant activity of the M.maderaspatana methanolic extract isshown in Figure 1 and compared with ascorbic acid. Both the extract andascorbic acid showed a linear increase in absorbance with increase in concen-tration. However, M.maderaspatana extract showed a significant change at20 to 80g/ml concentration and more powerful antioxidant activity thanascorbic acid by showing a higher absorbance than the corresponding ascor-bic acid concentration.

Reducing power assayThe total reducing power of M.maderaspatana methanolic extract comparedwith BHA is shown in Figure 2.Both the extract and BHA showed a linearincrease in the reducing activity in a dose dependent manner. The plantextract could reduce the Fe3+ ions, but had a lesser reductive activity thancorresponding concentration of BHA.

Cyclooxygenase inhibiting activityM. maderaspatana extract showed Cyclooxygenase inhibiting activity in adose dependent manner. The amount of extract which caused 50% inhibitionwas 8.42g. Under identical conditions 50 g of the standard phytochemicalsinhibited COX activity from 82% to 97% which is shown in Figure 3.

Lipoxygenase inhibiting activityThe M. maderaspatana extract inhibited 5-lipoxygenase in a dose dependentmanner (Figure 4). The amount of extract causing 50% inhibition of activitywas 4.63g. Under identical conditions 5g quercetin showed 58.9% inhibi-tion and 10 g of catechin showed 50.1% inhibition.

The M .maderaspatana extract inhibited 12-lipoxygenase and is shown inFigure 5. When 50 g each of M.maderaspatana extract and standardphytochemicals were assayed for their enzyme inhibition activity , the ex-tract gave the highest inhibition of 35.5% compared with quercetin whichshows only 21.7% inhibition.

Figure 1: Total antioxidant activity of methanolic extract of M. maderaspatana and ascorbic acid. The total antioxidant capacity was assayed based on the reduction of Mo (VI) to Mo (V) by the antioxidant compounds and formation of green phosphomolybdenum as described in the methods.

Figure 2: Reducing power activity of methanolic extract of M. maderaspatana and BHA. The total reducing power was assayed based on the ability of the antioxidant compounds to reduce Fe+++ to Fe++ as described in methods.

Figure 3: Cyclooxygenase inhibiting activity of methanolic extract of M. Maderaspatana and Catechin, Phloroglucinol, Quercetin and Ellagic acid. Cyclooxygenase activity was assayed by a fluorimetric method using Amplex Red reagent. Rat peritoneal macrophages were the source of enzyme and linoleic acid was used as the substrate. Plant extract and phytochemicals were tested for their ability to inhibit the formation of fluorescent resorufin from non fluorescent Amplex red reagent as described in methods.

Figure 4: 5-Lipoxygenase inhibiting activity of methanolic extract of M. Maderaspatana and Catechin, Phloroglucinol and Quercetin. 5-LOX activity was assayed using linoleic acid as substrate in rat peritoneal macrophages. Plant extract and phytochemicals were tested for their ability to inhibit formation of 5-HETE as described in methods.

Figure 5:12-Lipoxygenase inhibiting Activity of methanolic extract of M. Maderaspatana and Catechin, Phloroglucinol and Quercetin. 12-LOX activity was assayed using linoleic acid as substrate. Soybean extract was used as a source of the enzyme. Plant extract and phytochemicals were tested for their ability to inhibit formation of 12-HETE as described in methods.

6 0

7 0

8 0

9 0

1 0 0

Contr

ol

Phloro

glucin

ol 50ug

Catec

hin 50

ug

Querc

etin 50

ug

Mukia

50 ug

Per

cent

Act

ivit

y

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

contro

l

Mukia

5ug

Mukia

10ug

Ellagi

c acid

50ug

Catec

hin 50

ug

Phlor

ogluci

nol 50

ug

Querc

etin 50

ug

Per

cent

Act

ivity

- 0 . 1

0

0 . 1

0 . 2

0 . 3

0 . 4

0 . 5

0 . 6

0 . 7

0 . 8

0 . 9

0 2 0 4 0 6 0 8 0C o n c e n t r a t i o n g / m l

OD

at 7

00 n

m

B H AMukia

- 0 . 1

0

0 . 1

0 . 2

0 . 3

0 . 4

0 . 5

0 . 6

0 . 7

0 . 8

0 . 9

0 2 0 4 0 6 0 8 0 1 0 0 1 2 0C o n c e n t r a t i o n g / m l

OD

at 6

95 n

m

A s c o r b i c a c i dM u k i a

02 04 06 08 0

1 0 01 2 0

Contr

ol

Phlor

ogluci

nol 50

ug

Catech

in 10ug

Querc

etin 5u

g

Mukia

5ug

Mukia

15 ug

Per

cent

Act

ivit

yP

erce

nt A

ctiv

ity



3296-3303

Table 3: Radical scavenging activities of Mukia maderaspatana extractRadical scavenging assay Reference IC50 values(g/ml) of

Standards Standard Mukia extract

Hydroxyl radical BHA 37.6 22.9DPPH radical BHA 8.1 15.9Nitric oxide Ascorbic acid 155.7 269Superoxide Catechin 400 300

The radical scavenging assays were carried out using the methanolic extract of M.maderaspatana and compared with reference standards. The IC50 values were calculated fromlinearized plots of concentration versus activity.

Hydroxyl radical scavenging assayThe scavenging activity of M.maderaspatana extract on OH is shown inFigure 6 and compared with BHA. The IC50 value (Table 3) of scavengingeffect was 22.9g/ml and 37.6g/ml for the extract and standard BHA re-spectively. M.maderaspatana extract showed inhibition of OH formationduring incubation period and the percentage of inhibition was higher thanthat of BHA at all concentrations.

DPPH radical scavenging assayThe DPPH assay is based on the ability of DPPH, a stable free radical, todecolourize in the presence of antioxidants. The scavenging activity ofM.maderaspatana extract on DPPH is shown in Figure 7 and compared withBHA. The IC50 values (Table 3) of the standard BHA and extract was foundto be 8.1g/ml and 15.9g/ml respectively. The results show that BHA hadthe higher DPPH scavenging activity than the extract. The scavenging activ-ity was found to be concentration dependent.

Nitric oxide radical scavenging assayM. Maderaspatana extract decreased the amount of nitrite generated fromthe decomposition of sodium nitroprusside. In this assay, the suppression of

nitrite release may be attributed to a direct NO scavenging effect and isshown in Figure 8 compared with Ascorbic acid. The results show thatextract and reference compound, ascorbic acid had scavenging activity withIC50 values of 269.0g/ml and 155.7g/ml respectively (Table 3). The Ascor-bic acid showed higher NO scavenging activity than the extract.

Superoxide radical scavenging assayThe superoxide anion derived from dissolved oxygen by Phenazinemethosulphate/NADH coupling reaction reduces nitro blue tetrazolium. Fig-ure 9 shows the ability of M.maderaspatana and the standard catechin toquench superoxide radicals in this scavenging activity assay. As mentioned inTable 3, the extract as well as standard catechin showed the scavengingactivity with IC50 values of 300g/ml and 400g/ml respectively. The per-centage of inhibition by the extract was greater than that of catechin.

DISCUSSIONReactive oxygen species( ROS), such as superoxide anions, hydrogen perox-ide, and hydroxyl, nitric oxide and peroxynitrite radicals, play an importantrole in oxidative stress related to the pathogenesis of various disease.[32]

Most of the natural products contain abundant and potent antioxidants.They exert their beneficial effects through the additive action of severalchemical compounds acting at single or multiple target sites associated witha physiological process in contrast to synthetic antioxidants based upon asingle chemical. The antioxidant activity of the antioxidants have been attrib-uted to various mechanisms, among which are prevention of chain initiation,binding of transition metal ion catalysts, decomposition of peroxides, pre-vention of continued hydrogen abstraction, reductive capacity and radicalscavenging.[33]

Our phytochemical analysis (Table 1) is consistent with literature, although

Figure 6: Hydroxyl radical scavenging activity of methanolic extract of M. maderaspatana and BHA. The Hydroxyl radical was generated from H2O2 and reacted with deoxydibose. The degradation products of deoxyribose were reacted thiobarbituric acid. The ability of the antioxidant compounds was tested by their ability of scavenge OH radicals as described in the methods.

Figure 7: DPPH radical scavenging activity of methanolic extract of M. maderaspatana and BHA. The DPPH radical scavenging activity was determined by the ability of the antioxidant molecules to donate an electron or hydrogen radical to DPPH to make it a stable diamagnetic molecule as described in the methods.

Figure 8: Nitric oxide radical scavenging activity of methanolic extract of M. maderaspatana and Ascorbic acid. NO generated from sodium nitroprusside reacts with oxygen to produce nitrite ion, which is measured by the Griess reaction. The ability of the antioxidant molecule to prevent the formation of nitrite was used as a measure of nitric oxide scavenging activity as described in the methods.

01 02 03 04 05 06 07 08 09 0

0 2 0 0 400 600 800 1000 1200

Concentration g/ml

Per

cent

Inh

ibit

ion

CatechinMukia

Figure 9: Superoxide radical scavenging activity of methanolic extract of M. maderaspatana and Catechin. The superoxide anion scavenging activity was determined by the ability of the antioxidant molecules to decrease the formation of reduced NBT as described in the methods.

0

20

40

60

80

100

120

0 20 40 60 80 100Concentration g/ml

Perc

ent I

nhib

ition

BHAMukia

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

0 5 0 1 0 0 1 5 0 2 0 0 2 5 0C o n c e n t r a t i o n g / m l

Per

cent

Inh

ibit

ion

B H AMukia

0

2 0

4 0

6 0

8 0

1 0 0

0 2 0 0 4 0 0 6 0 0 8 0 0C o n c e n t r a t i o n g / m l

Perc

ent I

nhib

ition

A s c o r b i c A c i d

M u k i a



3296-3303

we have not identified the individual components, the class of moleculesreported in literature are shown to be present in the methanolic extract. In thetotal phenolics and flavonoids estimation our result show M. maderaspatanacontained significant amount of flavonoids and phenolics (Table 2). Phy-tochemical studies of the entire leaf extract of M. maderaspatana report thepresence of coumarins, phenolic compounds, flavonoids, steroid, triterpene,multiple glycosides (22,23-dihydrospinasterol3-O--D-glucoside),decosanoic acid and mixture of fatty acids.[34] The polyphenolic compoundsare major plant constituents having potential antioxidant activity mainly dueto their redox properties. The mechanism of action of flavonoids is throughscavenging or chelating process and phenolic contents are also very impor-tant plant constituents because of their scavenging ability due to their hy-droxyl groups.[1] Both of these compounds have good antioxidant potentialand their effects on human nutrition and health are considerable.

The total antioxidant activity of M.maderaspatana increased with increasingconcentration of the extract indicating the reduction of Mo (VI) to Mo (V)and subsequent formation of phosphomolybdate complex by donating anelectron. The higher reducing power shown by the methanolic extract incomparison with ascorbic acid(Figure 1) suggests that the methanolic extractis able to reduce water soluble oxidants and that it is a better anti oxidant thanascorbic acid. Antioxidant activity has been reported to go together with thedevelopment of reducing power and is a significant indicator of its potentialantioxidant activity. [35] For measurements of the reductive ability, we alsostudied the Fe3+ to Fe2+ transformation in the presence of a methanolic extractusing the method of Oyaizu. [23] The reducing capacity of M. maderaspatanamight be due to its hydrogen donating ability. The reducing power of M.maderaspatana increased in a concentration dependent manner, indicatingthat some compounds are both electron donors and could react with freeradicals to convert them into more stable products. They can also terminateradical chain reactions and this activity was comparable with that of BHA(Figure 2).

There are three main LOX enzymes that are known to be involved in theleukotriene pathway, 5-LOX, 12-LOX, and 15-LOX. Most attention hasbeen focused on 5-LOX, which appears to be the key enzyme when it comesto blocking leukotriene synthesis.[36] The direct 5-LO inhibitors can be sub-divided into three groups: redox-active compounds, iron-ligand inhibitorswith weak redox-active properties, and (non-redox) competitive 5- LO in-hibitors. Natural products which are 5-LO inhibitors, reduce the active siteiron thereby uncoupling the catalytic cycle of the enzyme. Thus, phenols,flavonoids, and coumarins, are efficient 5-LO inhibitors in vitro and in vivo.[37]

The key regulatory enzyme of the pathway of conversion of arachidonic acidto prostaglandin (PG) G2 and PGH2.is COX (PGH synthase). PGH2 issubsequently converted to a variety of eicosanoids that include PGE2, PGD2,PGF2a, PGI2, and thromboxane A2. Prostaglandins are formed by the oxida-tive cyclization of the central 5 carbons within 20 carbon polyunsaturatedfatty acids It is now well established that there are two distinct isoforms ofCOX, namely COX-1 and COX-2. Using the Fluorescent Activity Assayprovides a convenient fluorescence-based method for detecting COX-1 orCOX-2 activity in both crude cell lysates and purified enzyme. preparationIdeally, one would like to be able to inhibit both COX-2 and 5-LOX simulta-neously, to maximize anti-inflammatory effects.[38] Interestingly, theM.maderaspatana extract had both COX inhibitory activity and 5-LOXinhibitory activity higher than the standard phytochemicals(Figure 3and 4).The inhibition was dose dependent. Current understanding of the biologicalsignificance of these enzymes and products in humans health and disease isstill limited.

The hydroxyl radical has a short half-life and is the most reactive and damag-ing of ROS. It causes oxidative damage to DNA, lipids and proteins.[39] The

M.maderaspatana extract was investigated for its ability to scavenge OHradicals generated by Fenton reaction. As the methanolic extract or standardBHA is added to the Fenton reaction mixture, the hydroxyl radicals arescavenged and prevent the degradation of 2-deoxyribose. Like many otherfree radicals, OH can be neutralized if it is provided with hydrogen atoms.Ourresults shown in Figure 6 and Table 3, indicate that the scavenging activitiesof the methanolic extract was higher than that of BHA. This may be becauseof the presence of phenolic substances which can have active hydrogendonating ability of hydroxyl substitutions. DPPH is stable nitrogen centeredfree radical which can be effectively scavenged by antioxidants and can ac-cept an electron or hydrogen radical to become a stable diamagnetic mol-ecule.[40] It has been widely used for rapid evaluation of the antioxidantactivity of plant extracts relative to other methods. In its radical form,DPPHdisappears on reduction by methanolic extract resulting in a colourchange from purple to yellow. Figure 7 and Table 3 illustrate the effect of M.maderaspatana on DPPHscavenging, is thought to be due to their hydrogendonating ability which was comparable to that of BHA. In addition, theability to scavenge the DPPH radicals is related to the inhibition of lipidperoxidation since the first attack of the free radicals is on the membranelipids. Nitric oxide radicals play important roles in various types of in-flammatory conditions including juvenile diabetes, multiple sclerosis, arthri-tis and ulcerative colitis. [41]Nitric oxide radical scavenging study proved that M. maderaspatana extractis a potent scavenger of nitric oxide. The extract inhibits nitrite formation bycompeting with oxygen to react with nitric oxide directly and also inhibit itssynthesis. The scavenging activities of the extracts and ascorbic acid showedthat the nitric oxide scavenging activity of the former is comparable to that ofthe latter (Figure 8 and Table 3). Further, the lethal consequence of NOincreases significantly upon reaction with superoxide radical resulting in theformation of highly reactive peroxynitrite anion (ONOO-), especially itsprotonated form, peroxynitrous acid (ONOOH). ONOO- has added to thepathogenesis of diseases such as heart disease, Alzheimers disease, andatherosclerosis.Superoxides are produced from molecular oxygen due to oxi-dative enzymes [42] of body as well as via non enzymatic reaction such as autooxidation by catecholamines. In the present study, superoxide anion derivedfrom dissolved oxygen by PMS/NADH coupling reaction reduces NBT. Thefigure 9 and Table 3 shows the scavenging effect of M. maderaspatana. Thedecrease in absorbance at 560 nm with various concentration of extract indi-cates the consumption of superoxide anion in the reaction mixture. Superox-ide anions indirectly initiate the lipid oxidation as a result of superoxide andhydrogen peroxide, serving as precursors of singlet oxygen and OH. Inhibi-tion of superoxide generation by M. maderaspatana may be due to thepresence of flavonoids. Robak [43] reported that the antioxidant properties offlavonoids are effective primarily by the scavenging of superoxide anion. Inour study the superoxide radical scavenging activity of the extract was betterthan that of Catechin (Figure 9).

CONCLUSIONThe antioxidative data presented in this study demonstrates that M.maderaspatana exhibits free radical scavenging activity as well as a primaryantioxidant activity that reacts with free radicals, which may limit free radicaldamage occurring in the human body. The antioxidative effects of M.maderaspatana were accomplished in a dose-dependent manner. The vari-ous antioxidant properties of the extract may be attributed to strong hydro-gen donating ability and their effectiveness as scavengers of OH, superoxide,Nitric oxide, DPPH and other free radicals. Simultaneous inhibition of COXand LOX by extract suggests that it is potentially anti inflammatory. Thus,M. maderaspatana has a powerful antioxidant activity against various invitro oxidative systems and would probably be equally effective against invivo radicals and oxidants. Since M. maderaspatana is an edible plant it can



3296-3303

be a source of natural antioxidants and anti inflammatory molecules anduseful as potential food supplement.

ACKNOWLEDGEMENTSThe financial assistance from the Institution of Excellence (IOE) project ofthe University of Mysore is gratefully acknowledged. B.R.Srilatha is thank-ful to the IOE for the award of Fellowship.

REFERENCES1. Cook NC, Samman S, Flavonoids-chemistry, metabolism,

cardioprotective effects and dietary sources, Nutr Biochem, 7,1996, 66-76.

2. Pryor WA, Free radical reactions and their importance inbiochemical systems, Fed Proc, 32, 1973, 1862- 1869.

3. Adelman R, Saul RL, Ames B, Oxidative damage to DNA: relationto species metabolic rate and life-span, Proc Nal Acad Sci, 85,1998, 2706-2708.

4. Bergstrom S, Holman RT, Total conjugation of linoleic acid inoxidation with lipoxidase, Nature, 161(4080), 1948, 55. [PubMed: 18899663]

5. Mead, J. F., Alfin-Slater, R. B., Howton, D. R., and Popjak, G,Prostaglandins, thromboxanes, andprostacyclin, InLipids:Chemistry, Biochemistry, and Nutrition (Mead, J. F., ed),Plenum Press, New York. 1986, pp, 149-216.

6. Branen A, Toxicology and biochemistry of butylated hydroxyani-sole and butylated hydroxytoluene, J Am Oil Chem Soc, 52, 1975,59-63.

7. Sadzuka Y, Sugiyama T, Shimoi K, Kinae N, Hirota S, Protectiveeffect of flavonoids on doxorubicin induced cardiotoxicity, Toxi-cology Letters, 92,1997, 1-7.

8. Marchand LL, Cancer preventive effects of flavonoids: a review,Biomedicine and Pharmacotherapy 56, 2002, 296-301.

9. Yamaguchi M, Gao YH, Inhibitory effect of genistein on boneresorption in tissue culture, Biochemical Pharmacology, 55,1998, 71-76.

10. Thabrew MIRA, Jayatilaka PAPW, Perera DJB, Evaluation ofefficacy of Melothira madaraspatana in the alleviation of carbontetrachloride induced liver dysfunction, J Ethnopharmacol, 23,1988, 305-312.

11. Sinha BN, Sasnal D, Basu SP, Pharmacological studies on Melothriamaderaspatana, Fitoterapia, 68, 1997, 75-78.

12. Ramakrishnamacharya CH, Krishnaswamy MR, Rao RB,Vishwanathan S, Anti- inflammatory efficacy of Melothriamaderaspatana in active rheumatoid arthritis, Clin Rheumatol,15(2), 1996, 214-215.

13. Thabrew MI, De Silva KTD, Labadio RP, De Bio PAF, VanderBerg B,Immunomodulatory activity of three SriLankan medicinalplants used in hepatic disorders, J Ethanopharmacol, 33(1-2),1991,63-66.

14. Iman RA, Lakshmi Priya B, Chithra R, Shalini K, Sharon V,Chamundeeswari D, Vasantha J,et al, In vitro antiplatelet activityguided fractionation of aerial parts of Melothria maderaspatana,Indian J Pharm Sci, 68(5),2006, 668-670.

15. Hemamalini K, Varma VK, Antimicrobial activity of methanolicleaves extract of Melothria maderaspatana Linn,Pharmacologyonline, 3, 2007, 323-326.

16. Raja B, Kaviarasan K, Arjunan MM, Pugalendi KV,Effect ofMelothria Maderaspatana leaf-tea consumption on blood pres-sure, lipid profile, anthropometry, fibrinogen, bilirubin and albu-min levels in patients with hypertension, J Altern ComplementMed, 13(3),2007,349-354.

17. Kokate CK, Textbook of pharmacognosy, Nirali Publ, Pune, 1985,112.

18. Singleton VL, Rossi JA, Colorimetry of total phenolics with phos-phomolybdic- phosphotungstic acid reagents, Am J Eno Vitic,16,1965,144-158.

19. Chang C, Yang M, Wen H, Chem J, Estimation of total flavonoidcontent in propolis by two complementary colorimetric methods,J Food Drug Analysis, 10, 2002, 178-182.

20. Swain T, Hillis WE, The phenolic constituents of Prunusdomestica.I. The quantitative analysis of phenolic constitu-ents, J Sci Fd Agri, 10, 1995, 63-68.

21. Delcour JA, DeVarebeke DJ, A new colorimetric assay for fla-vonoids in Pilsner Beers, J Inst Brew, 91, 1985, 37-40.

22. Prieto P, Pineda M, Aguilar MM, Spectrophotometric quantitationof antioxidant capacity through the formation of aphosphomolybdenum complex: specific application to the deter-mination of vitamin E, Anal Biochem, 269,1999,337-341.

23. Oyaizu M, Studies on product of browning reaction preparedfrom glucose amine, Japan J Nutr, 44, 1986, 307-315.

24. Suzuki Y and Murachi T, The Occurrence of a Neutral Proteaseand Its Inhibitor in Rat Peritoneal Macrophages,J.Biochem,82,1977, 215-220.

25. Zhou, M., Diwu, Z., Panchuk-Voloshina, N., and Haugland, R, Astable nonfluorescent derivative of resorufin for the fluorometricdetermination of trace hydrogen peroxide: applications in detect-ing the activity of phagocyte NADPH oxidase and other oxidases,Anal. Biochem, 253, 1997, 162-168.

26. D. Aharony, R.L. Stein, Kinetic mechanism of guinea pig neutro-phil 5-lipoxygenase, J. Biol. Chem, 261, 1986, 1151211519.

27. Axelrod, B., Cheesebrough, T.M., & Laasko, S, Lipoxygenase fromsoybeans, Methods in Enzymology, 71, 1981, 441451.

28. Halliwell B, Gutteridge JMC, Arnoma OL, The deoxyribosemethod: A simple test tube assay for the determination of rateconstant for reaction of hydroxyl radical, AnalBiochem,165,1987,215-219

29. Brand Williams W, Cuvelier ME, Berset C, Use of free radicalmethod to evaluate antioxidant activity, Lebensmittel-Wissenschaftand Technologi, 1995, 28:25-30.

30. Green LC, Wanger DA, Glogowski J, Analysis of nitrate, nitriteand (15 N) Nitrate in biological fluids, Anal Biochem, 126, 1982,131-138.

31. Nishimiki M, Appaji N, Yagi K, The occurrence of superoxideanion in the reaction of reduced phenazine methosulphate andmolecular oxygen, Biochem Biophys Res Commun,46, 1972,849-854.

32. Finkel, T and Holbrook, N.-J, Oxidants, oxidative stress and thebiology of aging, Nature, 408, 2000, 239-247.

33. Oktay M,Gulcin I, Kufrevioiglu OI, Determination of in vitroantioxidant activity of fennel (Foeniculum vulgare) seed extracts,Lebensm Wiss Technol, Food Sci Technol, 36, 2003, 263-271.

34. Sinha BN, Sasmal D, Basu SP, Pharmacological studies on Melothriamaderaspatana,Fitoterapia,1, 1997,75-78.

35. Yen GC, Duh PD, Tsai CL, Relationship between antioxidant ac-tivity and maturity of peanut hulls, J Agric Food Chem, 41, 1993,67-70.

36. Smith W. L. and Murphy R. C, The eicosanoids: cyclooxygenase,

37. Yoshimoto T, Furukawa M, Yamamoto S, Horie T and Watanabe-

lipoxygenase and epoxygenase pathways D.E. Vzmcc and J.E.Vance (Eds.), Biochemistry of Lipids. Lipoproteiils apul Mem-branes (4th Edn), Elsevier Science B.V.2002, CHAPTER 13, pp341-371.



3296-3303

Source of support: Nil, Conflict of interest: None Declared

Kohno S, Flavonoids: potent inhibitors of arachidonate 5-lipoxygenase, Biochem Biophys Res Commun, 116,1983, 612-8.

38. Altavilla D, Squadrito F, Bitto A, Polito F, Burnett BP, Di StefanoV. and Minutoli L, Flavocoxid, a dual inhibitor of cyclooxygenaseand 5-lipoxygenase, blunts pro-inflammatory phenotype activa-tion in endotoxin-stimulated macrophages, Br J Pharmacol, 157,2009, 1410-8.

39. Spencer JPE, Jenner A, Aruoma OI, Intensive oxidative DNA dam-age promoted by L-DOPA and its metabolites implications forneurodegenerative disease, FEBS Lett, 353,1994,246-250.

40. Villano D, Fernandez-Pachona MS, Moyab ML, Troncosoa AM,Garcia- Parrilla MC, Radical scavenging ability of polyphenoliccompounds towards DPPH free radical,Talanta,71(Suppl1),2007,230-235.

41. Miller MJ, Sadowska-Krowicka H, Chotinaruemol S, Kakkis JL,Clark DA, Amelioration of chronic ileitis by nitric oxide synthaseinhibition, The JPharmac and Experi Thera, 264,1993,11-16.

42. Hemmani T, Parihar MS, Reactive oxygen species and oxidativeDNA damage, Ind J Physiol Pharmacol, 42, 1998, 440-444.

43. Catherine A,Rice E, Nicholas J, George P, Structureantioxidantactivity relationships of flavonoids and phenolic acids, Free RadicBiol Med, 20, 1996,933-956.

Copyright of Journal of Pharmacy Research is the property of Journal of Pharmacy Research and its contentmay not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's expresswritten permission. However, users may print, download, or email articles for individual use.

in vitro antioxidant and free radical scavenging activities.pdf

Documents