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Anti-Sickling Propensitiesof (L) Plant's ExtractsCarica
papaya
Meena Sahu, Devshree Verma, K.K. Harris*
Department of Zoology, Government DB Girls' Post
GraduateCollege, Raipur, 492001, Chhattisgarh, India
Keywords: Plantextracts, Stem, Leaf, Seed
StudyArea: Raipur, IndiaCoordinates: 28°30'00"N 53°33'38"E
This project was approved by the ethical committee of the
DBGirls' PostGraduate College, Raipur, C.G., India
Vol. 06h(1):11-15Year 2019
ambientSCIENCE
Ambient Science, 2019: Vol. 06h(1);
11-15DOI:10.21276/ambi.2019.06h.1.oa02
Sicklecell disease (SCD) isageneticdisorderand thediseaseis
incurable. The management process solely concentrateson providing
relief to the patient during the crisis stage ofthe disease. In
this context, phytochemicals present in theplants with antisickling
propensities are widely exploited asa cure. Various plants have
been reported to possessantisickling propensities by various
authors. The presentstudy attempts to investigate in vitro, the
antisicklingendeavors (sicklecell reversal and erythrocytic
inhibition toa sickled contour) of the leaves, seeds and stem
extracts of
L. in HbSS blood samples, using p-hydroxybenzoicacid and
phosphate buffersaline as positiveand negative controls. The
inhibition and reversal activitiesfor different concentrations
viz., 0.1 to 10.0 mg/ml ofextracts revealed maximum inhibition
activity (IA) of71.80% in L. leaves inaconcentrationof 1.0
mg/ml;seeds showed a IA of 68.50% in a concentration of 2.5mg/ml;
while the stems showed a IA of 68.33% in aconcentration of 10.0
mg/ml. On the other hand, maximumreversal activity (RA) of 72.43%
in Carica papaya L. leaves inaconcentrationof 10.0 mg/ml;
seedsshowed a RAof 65.96%in a concentration of 10.0 mg/ml; while
the stems showed aRAof 62.97% inaconcentrationof 10.0 mg/ml.
Carica papaya
C.papaya
Abstract
Introduction:Sickle cell disease (SCD) is known to be one of the
diseaseswrecking most parts of the globe without anydiscrimination
againstethnicor racial standards. Sickle celldisease includes those
that produce protuberant medicalexpressions as seen during sickle
cell anemia, sickle celldisease, sickle cell trait and an array of
other relatedhaemoglobinopathies (Hartwell 2000, Iyamu ,2003).
Pathophysiology of sickle cell anaemia, sickle celldisease, sickle
cell trait and an array of other relatedhaemoglobinopathies are now
known to the scientif iccommunity. Due to polymerization of the
sickled cells, thered cell membrane loses its functional abilities
which resultin loss of potassium and water and a corresponding gain
ofsodium ion. Increased intracellular free calcium occursduring
sickling (Brugnara, , 1993) resulting in a loss ofpotassium with
accompanying movements of chloride andwater. Small blood vessels
are blocked by the clumping ofsickled erythrocytes, averting blood
supply to variousorgans. The process of de-oxygenation in tissue
capillariescauses damage to its endothelium, leading to exudation
ofplasma into the surrounding soft tissue. This is
et al, et al.
et al.
characteristic of the soft tissue swelling seen in most
sicklecell diseasepatients (Olufunmilayo , 2010).
Various reports on the antisickling properties ofvarious plant
extract on human erythrocytes are availablewhich iswell tabulated
inTable-1.
Phytochemical analysis Preparation of Plant extracts: theleaves,
stems, and seeds were separately cut into small bits,and air dried
on shadow for about a fortnight. After dryingtheywere ground and
powdered, with 1 mm size by using agrinding machine before being
subjected tophytochemicalscreening. Four solvents, ethanol,
methanol andchloroform, and petroleum ether were used for
theextraction of different parts of the plants based on
theirincreasing polarity. Total 30g of the powdered leaves,
seeds,and stems of L. were extracted in differentsolvents in
Soxhlet apparatus of 250 ml. of each solventseparately concentrated
by slow evaporation process for 48hours (Harborne, 1973). The
obtained crude extracts werekept in the closed containers for
preliminary qualitativephytochemical analysis.
et al.
Carica papaya
Materialsand methods:
ORIGINAL ARTICLE
*Corresponding Author:
ISSN- 2348 5191 (Print) & 2348 8980 (Electronic)
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Qualitative Phytochemical Screening: the extracts of
eachpowdered parts of plants were used for phytochemical testsand
to identify the constituents, standard procedures werecarried out
(Trease & Evans, 1989; Sofowora, 1993). Thefollowing
phytochemicals: tannin, saponin, reducingsugar, alkaloid,
terpenoides, flavonoids, cardiac glycosidesanthraquinonesand
phenolswere tested as by following theprescribed methods (Trease
& Evans, 1989; Sofowora, 1993).
we used chemicals of analyticalgraded were methanol, petroleum
ether, phosphatebuffered saline tablets pH 7.2, liquid paraff in,
formalin,para-hydroxybenzoic acid, paraff in wax, EDTA bottles
anddistilled water forthisstudy.
the fresh leaves, stems and seeds
Antisickling activity:
Plant samplecollection:
of L. were simultaneously collected fromabundant farms,
cultivated farms and the open f ields inand around Raipur district.
After washing properly, theywere separately cut into small bits and
air dried in shadowfor about a fortnight. After drying they were
grinded andpowdered by using a grinding machine and sieved
throughawith 1 mm size mesh. Finally theywere preserved in
closedair-tightcontainers foranalysis.
200 g sample was extracted inSoxhlet apparatus which was
described by the method of 1with petroleum ether (60-80 C) and
aqueous- methanol(60-80 C) in 1:3 as solvents (Ogoda ., 2002).
Theprepared extracts were stored at 4oC in freeze in dried formand
used for the antisickling activity test. Varyingconcentrations have
been prepared from the dried extractsand used for the antisickling
assay which was varied from0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 5.0 and
10.0 mg/ml of leaves, seedsand stemof L.
the blood samples used inthe evaluation of the antisickling
activity of plants in thisstudy were collected from electrophoresis
conf irmed HbSSSCD patients belonging to the age-group 16 to 25
years, ofboth sexes and it was ensured that they were not taking
anyallopathic or ayurvedic medications. The samples weredrawn in
the presence of a qualif ied pathologist. A total of5.0 ml of fresh
blood samples were collected each time byway of vein-puncture in
EDTA (Ethylene di-amine tetraacetic acid) anticoagulant tubes and
mixed gently toprevent lysing of the red blood cells. In order to
conf irmtheirSS nature the above obtained blood samples were f
irstcharacterized by Hemoglobin electrophoresis on
celluloseacetategel. Blood sampleswerestored in ±4 C
temperature.
for inhibitory activity test 0.2ml HbSS sample was pipeted in
test tubes in duplicates.Following which, 0.2 ml of phosphate
buffered salinesolution and 0.2 ml of different concentration of
theextracts were added serially. Finally, the mixture wasoverlaid
with 1 ml liquid paraff in wax and incubated in athermo-stated
water bath at 37 C for about 4 hours. Afterincubation 0.6 ml of
freshly prepared 2% sodium meta-bi-sulphite solution was added
under liquid paraff in with thehelp of a syringe and these mixtures
were mixed by rollingthe test tubes between the palms of hand
carefully. Thesemixtureswere again incubated foraboutone and half
hoursin 37 C in a thermo-stated water bath. After incubation
theliquid paraff in wax was removed with the help of a
Pasteurpipetteand the resultant mixturewas f ixed in 3 ml of 5%
v/vbuffered formalin (Cyril-Olutayo , 2009).
for reversal of sicklederythrocytes activity tests, 0.2 ml of
blood sample waspipeted into test tubes in duplicates. Further, 0.2
ml ofphosphate buffered saline solution was added and
themixturewasoverlaid with 1 ml liquid paraff inwax. A total of
Carica papaya
et al
Carica papaya
et al.
Preparation of Extracts:
Collection of blood sample:
Inhibition of erythrocytes:
Reversal of sickled erythrocytes:
o
o
o
o
o
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Table-1: Plant Species used to recover Sickle Cell Anaemia
Species AuthorPlant part
LeafWhole plant
Root
Whole plant
Whole plant
Whole plant
Whole plantLeaf, seed, podSeed
Whole plantBarkWhole plantWhole plantWhole plant
Whole plant
Whole plant
Whole plant
Whole plant
LeavesWhole plantLeavesLeavesWholeplantFlowers, root
Hymenocardia acida et alXylopia aethiopicaMonodora
myristicaUvaria chamae
et al.,Carica papaya
et al.Sorghum bicolorPlumbago zeylanica et al.Uvaria
chamaeJusticia tenella
et al.J. gendarussa, J. insularisTrema orientalis et al.Garcinia
kola et al.Vigna unguiculata et alVigna subterraneanDetarium
microcarpum et alMorus alba et alNigerian legumes et alMorinda
lucida et alThuja occidentalis et al.Cucumis maximaC. Sativus, C.
LonatusAthospora platensis et al.Monodora myristicaHelianthous
annusDiclioptera calorata et alEuphorbia hirta,Sorghum bicolorFicus
sycomorus et al.Alchordia cordifoliaCalliandra portoricensis et
al.C. haematocephalaLypomia batalus et al.Ocimum basilicum et
al.Carica papaya et al.Moringa oleifera et al.Achillea
fragrantissimaSphaeranthus indicus et al.
Ziziphus jujube
Ibrahim ., 2007& Uwakwe &
Nwaoguikpe 2008(2 recipe)
Egunyomi 2009&
Ibraheem , 2010
& Adejumo , 2010
,Mpiana , 2010
Mpiana , 2011Adejumo , 2011
& Simeone ., 2012
Gbadamosi ., 2012Meselhy ., 2012Ojiako ., 2012Avaligbe .,
2012
, Nwaoguikpe ,; 2013
, Nwaichi , 2013,
, Mpiana ., 2013
, Alphonsine , 2014
, Amujoyegbe ,2014Mpiana , 2014Tshilanda , 2014Naiho ,
2015Nwaoguikpe , 2015Alabdallat, 2016Chopda ,2017
and
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http://www.caves.res.in/
0.6 ml freshly prepared 2% sodium meta-bi-sulphitesolution was
added under liquid paraff in and thesemixtures were mixed by
rolling the test tubes between thepalms of hand carefully, after
that they were incubated in athermo-stated water bath at 37 C for
1½ hr. After incubationdifferent concentration of the extracts were
added seriallyunder liquid paraff in wax and again it was incubated
for 6hours at 37 C in a thermo-stated water bath. Further,
theliquid paraff in wax was removed with a Pasteur pipette andthe
resultant mixturewas f ixed by theadditionof 3 ml of 5%v/v buffered
formalin (Cyril-Olutayo , 2009).
the f ixed cells were centrifuged at 4000rpm for 15 minutes and
the supernatants were decantedwith a capillary tube. Slides were
prepared from f ixed cellsafter the process of centrifugation and
were observed undera high power objective (x40 and x100) of
ResearchTrinocular Microscope (LABOMED VISION 2000) aboutfour
hundred (400) cells (both sickled and normalerythrocytes) were
counted and the percentage sickled cellswererecorded (Cyril-Olutayo
, 2009).
Qulitative phytochemical analysis of L.: theresults of the
qualitative analysis of L. arepresented in (Table-2).
.
o
o
et al.
et al.
Carica papayaC.papaya
Counting of cells:
1 2 3 1 2 3 1 2 3 1 2 3+ + + + - + + - + + - ++ + + + + + + + +
+ + ++ + + + + + + - + + - ++ + + + + + - + + + - ++ + + - - + + +
+ + + ++ + + + + + + + + + + ++ + + + + - + + + - + +- + + - + + -
+ + - + ++ + + + + + + + + + + +
Key: 1= ; 2= ; 3= ; + ( ); - ( )
Results:
Table 1:- Phytochemical constituents of different extractsof the
leaves, seeds and stems of LCarica papaya
Components Ethanolic Methanolic Choloroform Petro. ether
Leaves Seeds Stem Positive Negative
TaninsSaponinsRed. SugarsAlkaloidsTerpinoidsFlavonoidsCardiac
Glyc.Anthro quinonPhenols
Antisickling activity Inhibition of erythrocyte activityof
LC.papaya .: the inhibitory activities found inincreasing
percentage in Leaves, Seeds and Stems (Fig.-1).The inhibitory
activities for different concentrations ofextracts revealed maximum
inhibition activity (IA) of71.80% in leaves in a concentration of
1.0 mg/ml;seeds showed a IA of 68.50% in a concentration of
2.5mg/ml; while the stems showed a IA of 68.33% in aconcentration
of 10.0 mg/ml. Morphology of drepanocytes(Sickle cells) of the HbSS
blood of nontreated, treated withPBS (-ve control), PHBA (+ve
control) and differentconcentration of the extracts of the leaves,
seeds and stemextractof L. werepresented on (Plate- 2&3).
C.papaya
C. papaya
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Leaf Seed Stem
a
jk
f
bd e
g hj
a
mh
f ed
c
hkl
a
im l
ig
ed
c
Figure 1: Difference in Means± SE Inhibitionof sicklecell
invitroatvarious concentration of Leaf, Seed & Stemextracts Bar
having similar alphabets do not differ from eachotheratp
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http://www.caves.res.in/
Reversal ofSickled Erythrocyteactivityof L.C.papaya :the
reversal activities found in increasing percentage inLeaves, Seeds
and Stems (Fig.-2). The Reversal of sickledErythrocyte for
different concentrations of extractsrevealed maximum reversal
activity (RA) of 72.43% in
leaves in a concentration of 10.0 mg/ml; seedsshowed a RA of
65.96% in a concentration of 10.0 mg/ml;while the stems showed a RA
of 62.97% in a concentrationof 10.0 mg/ml. Morphologyof
drepanocytes (Sicklecells) ofthe HbSS blood treated with different
concentration of theextracts of the leaves, seeds and stem extract
of L.werepresented on (Plate-3).
phytochemical results from leaves of L.showed most of the
positive reactions for flavonoids,phenols, and alkaloids. Tannins
and saponins were alsopositive for most of the parts studied. It is
clear from theobtained results on antisickling assay, that
maximumantisickling/reversal activity were shown by the
partscontaining high percentages of phenols and alkaloids.Hence,
the management of SCD could be sought in thenumerous methods which
could prevent polymerization oferythrocytes into sickle cells. The
haemoglobin moleculewas found to resist polymerization and changing
its shapeinto a sickle by all or in association of one of the
followingconditions- (i) the propensity and prof iciency of
thebiomolecule to bind to the deoxygenated haemoglobinmolecules
(Abdulmalik ., 2005; Bianchi ., 2009) (ii)alteration of amino acids
which are responsible for thequaternarystructuresof haemoglobin
molecules includingtheir active and contact sites (Oyewole ., 2008)
(iii)offer consistency and stability to the haemoglobinmolecule
(Oyewole ., 2008; Ibraheem ., 2010;Chikezie, 2011). Therefore,
antioxidants present in theantisickling agents are essential and
important as they arecapable of attaining one or all of the
above-mentionedconditions. Furthermore, the results attained in
thesephytochemical and antisickling assays can provideimportant
data towards the classif ication of extractsaccording to their
total phytochemical content and
C.papaya
C. papaya
Carica papaya
et al et al
et al
et al et al
Discussion:
antisickling potential; for different individual parts
viz.,leaves, stem, and seeds. The results further support theview
that phytochemicals with antioxidant properties canwell
actaspotential antisickling agents.
We are thankful to Dr. Rekha Pandey, Principal, Govt. DB
Girls'PG College Raipur for providing necessary research facilities
andencouragement. We sincerely thank the University
GrantsCommission, UGC-CRO, Bhopal, for f inancial assistance in
theformof various minorresearch projects sanctioned to the
ZoologyDepartmentof Govt. DB Girls' PG College Raipur..
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Plate-3: Morphology of drepanocytes of the HbSS blood
treatedwith L. shows reversal effect on Leaf extract(a)(1.0 mg/ml)
Seed extract (b)(2.5 mg/ml) stemextract (c) (10.0 mg/ml)
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Ambient Science, 2019: Vol. 06h(1);
11-15DOI:10.21276/ambi.2019.06h.1.oa02ORIGINAL ARTICLE
Ambient Science (2019) Vol.-06h(1):p. 15
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