Available online at www.ijpsdr.comInternational Journal of
Pharmaceutical Sciences and Drug Research 2012; 4(2):
143-146143Research Article ISSN 0975-248XEvaluation of DPPH Radical
Scavenging Activity and Reducing Powerof Four Selected Medicinal
Plants and Their CombinationsP. Padmanabhan*, S. N.
JangleDepartment of Biochemistry, Rural Medical College,
Loni-413736, Ahmednagar, Maharashtra, IndiaABSTRACTReactive oxygen
species [ROS] cause oxidative damage to the tissues and protection
from such damages are provided byendogenous and exogenous
antioxidants. Plant based antioxidants are preferred due to the
multiple mechanisms of actionsand of the phytochemicals present in
them. 80% alcoholic extract of leaves of Aloe vera, Bacopa
monniera, Moringaoleifera and rhizome of Zingiber officinale was
tested individually and in combination in equal proportion of each
extractfor DPPH scavenging activity and reducing power. The results
indicate that the combination of the extract has better
DPPHscavenging action and reducing power compared to the individual
plant extract indicating synergistic and supra additiveeffect of
phytochemicals present in the extract.Keywords: Reactive oxygen
species [ROS], antioxidants, phytochemicals, DPPH scavenging
activity, reducing power,synergistic effects.INTRODUCTIONReactive
oxygen species [ROS], sometimes called as activeoxygen species, are
various forms of activated oxygen, whichinclude free radicals such
as superoxide ions (O2-.) andhydroxyl radicals (OH.) as well as
non-free radical speciessuch as hydrogen peroxide (H2O2). [1] These
ROS play animportant role in degenerative or pathological
processes,such as aging, cancers, coronary heart diseases,
Alzheimersdisease, neurodegenerative disorders,
atherosclerosis,cataracts and inflammations. [2] Living organisms
haveantioxidant defence systems that protects against
oxidativedamage by removal or repair of damaged molecules. [3]
Theterm antioxidant refers to the activity of numerousvitamins,
minerals and phytochemicals which provideprotection against the
damage caused by ROS. [4]Antioxidants interfere with the oxidative
processes byscavenging free radicals, chelating free catalytic
metals andby acting as electron donors. [5] The natural
antioxidantmechanisms maybe insufficient in variety of conditions
andhence dietary intake of antioxidant compounds are important.[6]
The therapeutic effects of several medicinal plants areusually
attributed to their antioxidant phytochemicals. It hasbeen
suggested that there is an inverse relationship betweendietary
intake of antioxidant rich foods and incidence of*Corresponding
author: Ms. P. Padmanabhan,Department of Biochemistry, Rural
Medical College, PravaraInstitute of Medical Sciences (Deemed
University), Loni-413736, Ahmednagar, Maharashtra, India; Tel.:
+91-9860494394; E-mail: [email protected] diseases.
[1] Plant based antioxidants are preferred tothe synthetic ones
because of their multiple mechanisms ofactions and non-toxic
nature. These facts have inspiredwidespread screening of plants for
possible medicinal andantioxidant properties; the isolation and
characterization ofdiverse phytochemicals and the utilization to
antioxidants ofnatural origin to prevent the diseases. [7]Aloe vera
(L.) Burm f. (Aloe barbadensis Miller) orcommonly called as khorpad
in Marathi; is a perennialsucculent xerophytes, which develops
water storage tissues intheir leaves to survive in dry areas of low
or erratic rainfall. Ithas been reported by several authors that
different fractionsof Aloe vera as well as unfractionated whole gel
haveantioxidant effects. Glutathione peroxidase,
superoxidedismutase enzyme and phenolic antioxidants were found
tobe present in Aloe vera gel which may be responsible for
itsantioxidant effects. [8]Bacopa monniera Linn (Scrophulariaceae)
commonly knownas brahmi; is a component of several popular drugs
ofAyurvedic system of medicine. The whole plant of brahmihas shown
presence of phytochemicals like alkaloids,saponins, D-mannitol,
betulic acid, -sitosterols and stigmasterols showing health
beneficial effects. [9]Zingiber officinale or ginger belongs to
Zingiberaceae family.The rhizomes of ginger have been used as
medicine fromVedic period and is called maha aushadhi means the
greatmedicine. [10] The antioxidant properties of
[6]-gingerol,which is very effective therapeutic agent present in
ginger,studied in both in-vitro and in-vivo against UV
induceOn-line disponibil la www.ijpsdr.comInternational Journal of
Pharmaceutical Sciences i de cercetare de droguri 2012; 4 (2):
143-146143Cercetare Articolul ISSN 0975-248XEvaluarea DPPH
eliminrii radicalului de activitate i reducerea puteriide patru
Plante medicinale selectate i combinaii ale acestoraP. Padmanabhan
*, S. N. ceartDepartamentul de Biochimie, Rural Medical College,
Loni-413736, Ahmednagar, Maharashtra, IndiaREZUMATSpecii reactive
de oxigen [ROS] daune cauza oxidativ la esuturile i protecia
mpotriva unor astfel de daune sunt furnizate de ctreendogene i
exogene antioxidani. Antioxidani pe baz de plante sunt preferate
datorit mecanisme multiple de actiunii a fitochimicale prezente n
ele. Extract alcoolic 80% din frunze de aloe vera, Bacopa monniera,
Moringaoleifera i rizom de Zingiber officinale a fost testat
individual i n combinaie n proporie egal de fiecare extractpentru
activitatea de eliminare DPPH i reducerea puterii. Rezultatele
indic faptul c ansamblul de extract are mai bine DPPHbaleiaj aciune
i reducerea energie n comparaie cu extractul instalaiei individuale
care indic aditiv sinergic i supraefect de fitochimicale prezente n
extractul.Cuvinte cheie: specii de oxigen reactive [ROS],
antioxidanti, fitochimicale, activitatea de eliminare DPPH,
reducerea puterii,efecte sinergice.INTRODUCERESpecii reactive de
oxigen [ROS], uneori numit ca activspecii de oxigen, sunt diferite
forme de oxigen activat, careinclud radicalii liberi, cum ar fi
ionii de superoxid (O2-.) iRadicalii hidroxil (OH.), precum i
specii de radicali liberi non-cum ar fi peroxid de hidrogen (H2O2).
[1] Aceste ROS joac unrol important n procesele degenerative sau
patologice,cum ar fi mbtrnirea, cancer, boli coronariene, boala
AlzheimerBoala, tulburri neurodegenerative, ateroscleroza,cataracta
si inflamatiile. [2] organisme vii ausisteme de aprare antioxidante
care protejeaza impotriva oxidativdaune prin eliminarea sau
repararea de molecule deteriorate. [3]Termenul "antioxidant" se
refer la activitatea de numeroasevitamine, minerale i fitochimicale
care furnizeazprotecie mpotriva daunelor cauzate de ROS.
[4]Antioxidantii interfera cu procesele oxidative decoridoarele
radicalilor liberi, chelatori de metale catalitice libere iacionnd
n calitate de donori de electroni. [5] natural antioxidantmecanisme
poate insuficiente n varietate de condiii iprin urmare, aportul
alimentar de compusi antioxidanti sunt importante.[6] Efectele
terapeutice ale multor plante medicinale suntde obicei atribuita
fitochimicale lor antioxidante. AreSa sugerat c exist o relaie
invers ntreaportul alimentar de alimente bogate antioxidante i
incidena* Corespondent autor: Dna P. Padmanabhan,Departamentul de
Biochimie, Rural Medical College, PravaraInstitutul de Stiinte
Medicale (Universitatea estimate), Loni-413736, Ahmednagar,
Maharashtra, India; Tel .: + 91-9860494394; E-mail:
[email protected] umane. [1] antioxidanti plante pe
baza sunt preferatecele sintetice din cauza mai multor mecanisme
lor deaciuni i natura non-toxic. Aceste fapte au
inspiratscreening-ul pe scara larga de plante pentru posibile
medicinale iproprieti antioxidante; izolarea i
caracterizareadiverse fitochimicale si antioxidanti utilizarea a
deorigine natural pentru a preveni bolile. [7]Aloe vera (L.) Burm
f. (Aloe barbadensis Miller) saude obicei numit ca "khorpad" n
Marathi; este o planta perenaxerofite suculente, care dezvolta
tesuturi de stocare a apei nfrunzele lor de a supravieui n zonele
uscate de precipitaii reduse sau neregulat. Aceastaa fost raportat
de mai muli autori care fraciuni diferitede aloe vera, precum i
ntregul gel nefractionata auefecte antioxidante. Glutation
peroxidaza, superoxidsuperoxid enzim i fenolici antioxidanti s-au
gsit lafi prezent n aloe vera gel care poate fi responsabil pentru
eiefecte antioxidante. [8]Bacopa monniera Linn (Scrophulariaceae)
denumitca "Brahmi"; este o component a multor medicamente cunoscute
deSistem de medicina ayurvedica. Planta ntreag de Brahmiare prezen
prezentat de fitochimicale ca alcaloizi,saponine, D-manitol, acid
betulic, p-sitosterols i stigmatsteroli care prezint efecte
benefice pentru sntate. [9]Officinale Zingiber sau ghimbir aparine
familiei Zingiberaceae.Cele Rizomi de ghimbir au fost folosite ca
medicament de laEpoca vedic i se numete '' Maha aushadhi "nseamn
cel Maremedicament. [10] De proprieti antioxidante [6]
-gingerol,care este foarte eficient agent terapeutic prezent n
ghimbir,studiat n ambele in vitro si in vivo impotriva UV
inducePadmanabhan et al. / Evaluation of DPPH Radical Scavenging
Activity and Reducing Power..IJPSDR April-June, 2012, Vol 4, Issue
2 (143-146) 144Moringa oleifera Lam commonly known as
drumstick,belongs to Moringaceae family. The derivatives of
caffeic, pcoumaricand ferulic acids are dominant
phenolicconstituents which are present in Moringa oleifera
leavesextract. [12]The different plant extracts will have different
modes ofaction for curing diseases and in mixture form may
exhibitenhanced activity than that of individual plants, which
isknown as synergistic action. A particular principle in thepure
form may have only a fraction of the pharmacologicalactivity than
it has in its plant matrix. This highlights theimportance of using
the plant as a whole or a mixture ofplants for treating a disease.
[13] The objectives of the presentstudy were to quantify total
phenolics, flavonoids andflavonols in the combination of the four
medicinal plantextracts selected; that included leaves of Aloe
vera, Bacopamonniera, Moringa oleifera and rhizome of
Zingiberofficinale. It was also aimed to observe the
antioxidantproperties in terms of DPPH scavenging activity
andreducing power of the individual plant extracts and
theircombinations for existences of any synergistic property of
thefour medicinal plant extracts selected.MATERIALS AND
METHODSLeaves of Aloe vera, Bacopa monniera, Moringa oleifera
andrhizome of Zingiber officinale were collected from Loni
andadjoining areas, Maharashtra. These four individual plantswere
identified and authenticated by a Professor of Botany,Loni. The
four medicinal plants were shade dried andpowdered. The selected
part of the medicinal plants wasindividually extracted in 80%
ethanol by hot extraction inSoxhlet apparatus till colourless
solvent was obtained.Individual extracts obtained were allowed to
dry till constantweight was obtained. The percentage yield of
extract of eachplant was Aloe vera 28.62%, Bacopa monniera
16.18%,Moringa oleifera 14.90% and Zingiber officinale
12.69%.Concentration of combination of herbal
preparationCombination of plant extracts was prepared by mixing
25mg each of individual extract and dissolved in 10 mlmethanol
(that is 100mg/10ml), boiled and cooled. It was,then centrifuged at
2500 rpm for 10 minutes. The supernatantthus obtained was named as
Herbal Preparation (HP-4) andthis combination was used in further
experiments.Phytochemical AnalysisThe herbal preparation was
subjected to preliminaryphytochemical analysis using standard
procedures to estimatethe phytochemicals present.Estimation of
total phenolic compoundsTotal phenolic content was determined by
the Folin Ciocalteumethod by Folin et al 1927. [14] To 0.5 ml of
1-5 mg/ml ofherbal preparation made up with 0.5 ml of distilled
water, 0.5ml of Folin Ciocalteu reagent was added and gently
mixed.After 2 minutes 0.5 ml of 100mg/ml sodium carbonate wasadded.
The contents were mixed and allowed to stand for 2hours. The
optical density of the blue coloured samples wasmeasured at 765 nm
spectrophotometrically. Standard gallicacid of concentration
100-500g/ml was used. Theconcentration of total phenolics is
expressed as milligram ofgallic acid /g of mixture. All
determinations were carried outin triplicate.Estimation of
flavonoidsThe method used by Chang et al 2002 [15] with
slightmodifications in total volume of reagents used; was
followedfor estimation of flavonoids. 0.5 ml of concentration
100-500g/ml of herbal preparation was mixed with 1 mlaluminium
trichloride in ethanol (20g/l) and diluted withethanol to 25 ml.
The absorbance was read after 40 minutesincubation at 37C
spectrophotometrically at 415nm. Rutin (acitrus flavonoids
glycoside) of concentration 0.5mg/ml, 1.0mg/ml, 1.5 mg/ml, 2.0
mg/ml and 2.5 mg/ml was used as areference compound and absorbance
was measured under thesame conditions. All determinations were
carried intriplicate. The amount of flavonoids in herbal
preparationwas calculated as milligram of rutin/g of
mixture.Estimation of flavonolsThe content of flavonols was
determined by the method ofYermakov et al 1987 [16] with slight
modifications likereduction in total volume of reagents used. 0.05
ml of variousconcentrations (100-500g) was treated with 1ml of
2%aluminium trichloride in ethanol and 1ml of 5% sodiumacetate. The
absorption was read at 400nm was read after 2.5hours at 37C. The
same procedure was carried out for 2mlof reference compound rutin
for concentration 0.2mg/ml, 0.4mg/ml, 0.6 mg/ml, 0.8 mg/ml and 1.0
mg/ml. Alldeterminations were carried out in triplicate .The
content offlavonols was calculated in terms of milligram of rutin
/g ofmixture.Method for DPPH radical scavenging assayRadical
scavenging activity of plant extracts against stable 2,2 diphenyl 2
picryl hydrazyl hydrate (DPPH) was determinedby the slightly
modified method of Brand-Williams et al1995. [17] DPPH reacts with
an antioxidant compound, whichcan donate hydrogen, and reduce DPPH.
The change incolour (from deep violet to light yellow) was measured
at517 nm on a UV visible light spectrophotometer. Thesolution of
DPPH in methanol 6 10-5 M was prepared freshdaily before UV
measurements. Three ml of this solution wasmixed with 100
microgram/ml concentration of individualplant extracts as well as
herbal preparation. The sampleswere kept in the dark for 15 minutes
at room temperature andthe decrease in absorbance was measured. The
experimentwas carried out in triplicate. Radical scavenging
activity wascalculated by the following formula.% Inhibition = [(A
B A A)/A B] 100Where A B = absorption of blank sample (t= 0 min)A A
= absorption of test extract solution (t=15 mins) [18]Table 1:
Preliminary phytochemical analysisPhytochemicals ContentsTotal
Phenolic compounds (mg gallic acid/g) 29.53 0.42Flavonoids (mg
rutin/g) 24.05 0.57Flavonols (mg rutin/g) 17.9 0.24Table 2: DPPH
scavenging activityS. No Concentration (g/ml) % Inhibition*Aloe
vera 100 38.47 1.94Bacopa monniera 100 37.98 0.77Moringa oleifera
100 38.28 3.26Zingiber officinale 100 43.02 3.47Herbal Preparation
100 63.75 0.73**Std BHT 100 44.70 11.8Std Vitamin C 100 80.38
0.05*Mean SD of three determinations. **Statistically significant
(p
