International Conference on Global Trends in Engineering, Technology and Management (ICGTETM-2016)
Studies on Biological Efficacy of Various Leaf Extracts of
Carica Papaya L
Bhushan Kavimandan
Assistant Professor, Department of Biochemistry, Moolji Jaitha College, Jalgaon (MS) – 425001
Mrinal Saraf
Research Scholar, Department of Biochemistry, Moolji Jaitha College, Jalgaon (MS) – 425001
ABSTRACT:
Carica papaya L. is one of the tradinally important medicinal plant belongs to the family Caricaceae.
Phytochemical profiling of various organic and water extracts of 15 days old leaves depicted the presence of
various secondary metabolites like alkaloids, flavonoids, phenolic compounds etc. Remarkable antioxidant capacity
was observed in methanolic extract whereas maximum total phenolic compounds were present in water extract.
Antimicrobial activity against E. coli was observed in ethanolic extract with zone of inhibition of diameter 22 mm.
Partial purification of alkaloids was also done by column chromatography and method given by Osuagwu and
maximum yield obtained in dry powder.
KEYWORDS: antimicrobial activity, Carica papaya, secondary metabolites
I. INTRODUCTION:
Carica papaya L. belongs to the plant family
Caricaceae. A lot of work has been carried out on
plant parts like fruits, seeds and roots, indicating the
presence of biologically active compounds [21].
Traditionally leaves of Carica papaya have been
used for treatment of a wide range of ailments, like in
treatment
of
malaria,
dengue,
jaundice,
immunomodulatory and antiviral activity [27]. The
fruit, leaves and latex obtained from papaya plant are
used medicinally and for various other purposes.
Papain, extracted from fruit and stem latex is used in
brewing, wine making, textile and tanning industries
[7]. Study conducted by University of Florida
researchers had documented papaya’s powerful
anticancer properties and impact on numerous labgrown-tumors [22]
Proximate analysis of the plant parts showed that all
the macronutrients were present, with carbohydrate
being the most abundant in C. papaya. Vitamins A,
C, B12 and Folic acid were present but are void of
Vitamin E. Papaya leaf extract contained only
magnesium, the other metals tested were not detected
[13]. The study of the Carica papaya leaves of semi
arid zone of kutch region showed that it is rich in
elements like calcium, magnesium, sodium,
potassium, chlorine, and lithium with comparison to
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other reported analysis of other regions [26]. This is
in contradiction to above analysis and numbers of
minerals present in the leaf need to be explored. The
screening of leaves at all three stages (green, yellow
and brown) of Carica papaya had shown richness in
vitamins like thiamine, riboflavin, ascorbic acid.
Similarly minerals like calcium, magnesium, sodium,
potassium, manganese and iron were also detected at
considerable amount. The Phytochemical analysis of
the leaves showed that the leaves contained saponins,
cardiac glycosides and alkaloids [4]. The fruit, seed
and leaf of Carica papaya were found to be rich in
proteins. All the three parts were rich in not only
minerals (calcium, magnesium, phosphorous, and
iron) but also vitamins (vitamin C, niacin, thiamine,
riboflavin, and beta-carotene). Thus these nutrients in
the plant can turn out essential tool in growth of
humans and animals [8].
The qualitative phytochemical analysis revealed the
presence of all the phytochemical including
glycosides, alkaloids, saponins, flavonoids, proteins
except steroids and tannins in the leaf of papaya [23].
LCMS analysis followed by integrated library search
identified
21constituents
which
included
pharmacologically
active
phyto
compounds,
alkaloids, phenolics, flavonoids and also, amino acids
in Carica papaya [1].
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Methanolic and Ethyl acetate extract of leaf have
shown good activity against C.tropicalis, B.cereus
and E.coli. Acetone was more effective against
A.flavous, A.niger, B.cereus and P.Aereginosa.
Chloroform extract was found effective against
A.flavous, C.tropilcalis, M.leteus and E.coli. and
petroleum ether and n-Hexane were effective against
S.Aureus and not effective against fungal strains. It
has been proposed that Phytochemical compounds
such as tannin, coagulate the wall proteins, saponins,
facilitates the entry of toxic material or leakage of
vital constituents from the cell. Thus blocks the
growth [5]. Hot aqueous extract of ripe peel of
Carica papaya had shown best antibacterial activity
forming a zone of inhibition of 13 mm against the
Escherchia coli, which was equal to the zone of
inhibition formed by the standard antibiotic
Tetracycline (500 μg/ml). 80 % methanol extract of
unripe pulp shows an average zone of inhibition of 18
mm against the Candida albicans, which was nearly
equal to the zone of inhibition of Fluconazole [12].
Study revealed very significant antimicrobial activity
with the extracts of Carica papaya demonstrating
broad spectrum of activity against both bacteria (S.
pyogenes, E. coli, K. pneumonia, K. oxytocin, E.
faecalis, P.aeruginosa, P. vulgaris and S. aureus) and
fungi (A. flavus, T. metagrophytes and C. albicans)
[16]. The leaf extracts of Carica papaya in ethanol,
Chloroform, Acetone showed the positive inhibitory
effects in for the E.coli and salmonella sps. The
antimicrobial activities of these plants parts show
multidrug resistance against the pathogenic bacteria
as claimed by traditional healers. Much more
research is needed for the value added food
preservative agents for selected microbes can be
investigated [3].
The organic extracts of Carica papaya were more
effective than aqueous extracts and the ethyl acetate
extracts demonstrated the highest activity [15]. The
result of phytochemical analysis of leaf extracts of C.
papaya revealed the presence of Alkaloids,
Flavonoids, Saponins, Tannins. The antimicrobial
activity of the plant showed that the plant had a broad
spectrum activity against the test isolate with varying
zones of inhibitions ranging from 16.0mm, 18.4,
15.0, 16.5, and 13.0 mm for S. aureus, E. coli, S.
typhi, P. aeroginosa, and C. albican respectively at
100% concentration of the ethanolic extract [14]. C.
papaya in organic extracts such as petroleum ether
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and 1% HCl , were more effective than C. Papaya in
aqueous extracts like water. This may be due to the
fact that there is better solubility of the active
components in organic solvents [17].
The highest antioxidant activity through β-carotene
bleaching assay was observed in unripe fruit followed
by young leaves, ripe fruit and the seed of Carica
papaya. On other hand, young leaves exhibited a
significant higher scavenging effect compared to
others [6]. Flavonoids, glycosides and cardiac
glycosides found in the extracts are suggestive of
their antioxidant property. Flavonoids, glycosides are
reported to be antioxidants [10]. The antioxidant
activity of the plants of Carica papaya can be
resulted from the activity of various phytochemicals
present in the plant. However, not all the
phytochemicals are responsible for the antioxidant
activity, which means that even if a plant tested
positive for most of the phytochemicals, it is not
necessarily that it could show the highest antioxidant
activity. Thus it is important to identify which
phytochemicals are responsible for the antioxidant
activity [20].
Super oxide can be decomposed to form stronger
oxidative species such as singlet oxygen and
hydroxyl radicals, is very harmful to the cellular
components in a biological system. The super oxide
scavenging activity of Carica papaya leaf extract was
increased markedly with the increase of
concentration. The extract had important super oxide
scavenging activity [25]. The secondary metabolites
such as phenolics and flavonoids from plants have
been reported to be potent free radical scavengers.
They are found in all parts of plants such as leaves,
fruits, seeds, roots and bark [9]. The phenolic
content was in the order of leaves > unripe papaya >
ripe papaya > seed. HPLC analysis showed that
papaya leaves exhibited the highest ascorbic acid and
β-carotene content, while the seeds had the highest
Vitamin E content.
II. MATERIALS AND METHODS:
2.1 SAMPLE PREPARATION:
Fifteen days old leaves of papaya plant of breed
“Taiwan 786” were shade dried, powdered and used
for extraction. 25 grams powdered plant materials
(leaves) were soaked separately with 250 ml of each
of the ethanol, methanol, n-hexane and acetone, and
extracted in a soxhlet apparatus for 6 hrs at boiling
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point until complete extraction of the materials.
Extracts were concentrated at room temperature in
order to reduce the volume. In distilled water the
same was soaked separately with 250 ml and
macerated for 6 hours with some intervals. At the end
of 6 hrs, each extract was filtered through Whatman
No.1 filter paper and filtrates were concentrated at
room temperature in order to reduce the volume.
These extracts were used for further analyses.
2.2 PHYTOCHEMICAL PROFILING:
For establishing phytochemical profile of the
extracts, qualitative phytochemical analysis was
performed by using standard procedure with slight
modifications given by M. Begum [13]. The tests
were
for
presence/absence
of
alkaloids,
carbohydrates, proteins, fats, saponins, flavonoids,
phenolic contents etc.
2.3 BIOLOGICAL ACTIVITIES:
A) TOTAL POLYPHENOLIC CONTENT:
Determination of total polyphenolic content (TPC)
was done as described by Irondi [11] with slight
modifications. TPC was expressed as mg of gallic
acid equivalents (GAE) per gram of extract.
B) TOTAL ANTIOXIDANT CAPACITY:
Determination of total antioxidant capacity (TAC)
extracts of selected medicinal plants was evaluated
by phospho-molybdenum method according to the
procedure of Prieto [19]. Total antioxidant capacity
was expressed as mg of ascorbic acid equivalents per
gram of extracts.
C) ANTIMICROBIAL ACTIVITY:
Determination of the antimicrobial activity of papaya
leaf extract was done by paper disc diffusion method
with slight modifications using protocol given by
Aneja [2].
2.4
PARTIAL
PURIFICATION
OF
ALKALOIDS:
I) METHOD GIVEN BY OSUAGWU:
From the extracts alkaloids were obtained by the
method given by Osuagwu [18] with slight changes.
Five g of the dried extract was mixed with 200 ml of
10 % acetic acid in ethanol. The mixture was covered
and allowed to stand for 4 hours. This was filtered
and the filtrate was concentrated on a water bath until
it reaches to one-quarter of the original volume.
Concentrated Ammonium hydroxide was added drop
wise to the filtrate until the precipitation was
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complete. The whole solution was allowed to settle.
Precipitate was collected and washed with dilute
Ammonium hydroxide and then filtered. The residue
was the alkaloid, which was dried at 60oC for 30 min.
The precipitate was tested for presence/absence of
alkaloids.
II) COLUMN CHROMATOGRAPHY:
Activated silica was packed in the column and the
chloroform concentrated water extract was loaded
and separation of the mixture was achieved using
chloroform : acetone (40:60) as mobile phase. Forty
fractions of 20 ml each were collected; further
concentrated and subjected to paper chromatography
for detection of alkaloids using Dragendorff reagent
[24].
III. RESULTS:
3.1 PHYTOCHEMICAL PROFILING:
Phytochemical profiling of various extracts of C.
papaya had shown the presence alkaloids,
carbohydrates, saponings, proteins, fats, phenolic
compounds and flavonoids. Similar findings are also
mentioned by P.B. Ayoola [4], S. K. Sherwani [23],
Akhila [1], C. Baskaran, et al [5]. Alkaloids were
detected in almost all extracts whereas other
metabolites were detected in some extracts while
were absent in other. Table No. 1 shows the
phytochemical profiling of the extracts.
Table No. 1:
3.2 TOTAL PHENOLIC CONTENT:
Total phenolic content of various extracts are
depicted in the graph no. 2. Maximum phenolic
content was found in water extract whereas minimum
in n-hexane extract. Some results of total phenolic
content are not in agreement with qualitative
phytochemical profiling. The most probable reason is
low
sensitivity
of
qualitative
tests.
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3.5
PARTIAL
PURIFICATION
OF
ALKALOIDS:
I) OSUAGWU METHOD:
Partial purification of alkaloids was carried out by
two methods. The percentage of alkaloids obtained
by the method given by Osuagwu (2013) in various
extracts and dry powder of C. papaya leaves are
graph No. 6. Maximum yield was obtained in dry
powder followed by methanol and water extracts.
3.3 ANTI-OXIDANT CAPACITY:
Graph no. 4 shows the anti-oxidant capacity of
various extracts of C. papaya leaves. Maximum antioxidant capacity was observed in ethanolic extract
while in water extract, it was minimum.
II) COLUMN CHROMATOGRAPHY:
Partial purification of alkaloids was also carried out
by
column
chromatography
followed
by
identification using paper chromatography. Alkaloids
were found to be present in fraction No. 25, 26, 28,
29.
Figure No. 1
3.4 ANTIMICROBIAL ACTIVITY:
Graph No. 5 shows antimicrobial activity of various
extracts against E. coli. Maximum activity was seen
in ethanolic extract while minimum activity was
noted in water extract.
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IV. DISCUSSION:
Carica papaya is one of the important medicinal
plants. Its therapeutic value is because of various
chemical constituents present in it. Various biological
activities of C. papaya leaves extracts are attributed
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to phytochemical constituents. Secondary metabolites
like alkaloids, flavonoids, phenolic compounds etc
are responsible for various biological activities like
antioxidant, antimicrobial and so on. The presence of
such metabolites in the extracts also indicates the
same. The super oxide scavenging activity of Carica
papaya leaf extract was mentioned by Srikanth et al
[25]. The secondary metabolites such as phenolics
and flavonoids from plants have been reported to be
potent free radical scavengers. They have been
reported in all parts of plants such as leaves, fruits,
seeds, roots and bark by Ifesan [9].
C. Baskaran [5] reported antimicrobial activity of C.
papaya leaf extracts against various species of
bacteria and fungi. It has been proposed that
Phytochemical compounds such as tannin, coagulate
the wall proteins, saponins, facilitates the entry of
toxic material or leakage of vital constituents from
the cell. Thus blocks the growth of microorganisms.
Khan [12] got antimicrobial activity against
Escherchia coli and Candida albicans using hot
aqueous extract of ripe peel and 80 % methanol
extract of unripe pulp of Carica papaya respectively.
Very significant antimicrobial activity with the
extracts of Carica papaya against both bacteria (S.
pyogenes, E. coli, K. pneumonia, K. oxytocin, E.
faecalis, P.aeruginosa, P. vulgaris and S. aureus) and
fungi (A. flavus, T. metagrophytes and C. albicans)
was demonstrated by Okunola [16].
The present research work has shown phytochemical
composition, antimicrobial and antioxidant activity of
Carica papaya leaves extracts. Varied antimicrobial
and antioxidant activity by various extracts suggest
the varying degree of efficacy and different
phytochemical composition of the extracts.
Antimicrobial and antioxidant activity shown by
extracts can be attributed to the potential biocomponents present in them. Further studies are
essential
for
isolation,
purification
and
characterization of potential bio-components and
their exploitation in related fields.
North Maharashtra University, Jalgaon for funding
the current research project.
References:
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Acknowledgement:
The authors are very grateful to the Vice-Chancellor
Research Motivation Scheme (VCRMS) North
Maharashtra Univeristy, Jalgaon and Assistance for S
& T application through university systems of
RGS&TC, government of Maharashtra, through
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Table No. 1: Phytochemical Profiling of the extracts
Test
n-Hexane
Methanol
Water
1] Alkaloids
a.Mayers Test
+
+
b. Wagner’s Test
+
+
+
2] Carbohydrates
a.Fehlings Test
+
b.Benedicts Test
+
+
+
+
3] Saponing (foam test)
4] Protein & Amino Acids
a.Biuert Test
+
b.Ninhydrin Test
+
5] Fixed oils &Fats
a. Spots Test
+
+
+
6] Phenolic Compounds &
Tannins
a. Ferric Chloride Test
b. Lead Acetate Test
+
+
7] Flavonoids
a. Alkaline Reagent Test
+
+ presence; - absence
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Ethanol
Acetone
+
+
+
+
+
+
+
+
+
-
+
+
+
+
-
+
-
+
+
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Figure No. 1
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