3700

Advances in Environmental Biology, 5(12): 3700-3709, 2011

ISSN 1995-0756

This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE
 
Phytochemical, toxicity, antimicrobial and antioxidant screening of leaf extracts of
Peperomia pellucida from Nigeria

Ganiyat K.Oloyede, Patricia A. Onocha and Bamidele B. Olaniran

Na tural pro du ct s/ Medic inal Ch e mis t r y Uni t , Depa rt ment of Ch e mis t r y , Univ er sit y of
Ibadan , Nige ria .

Ga niyat K. Oloy ede, Pat ri cia A. On o cha and Bamidele B. Olaniran; Phytochemical,
toxicity, antimicrobial and antioxidant screening of leaf extracts of Peperomia pellucida from Nigeria

ABSTRACT

Peperomia pellucida (Piperaceae), known as Shiny bush is a herbaceous plant with ethno medical uses
which include anti-inflammatory and analgesic properties. Air dried leaves of the plant was extracted using
Soxhlet extractor to give the methanol extract which was then partitioned successively in n-hexane, ethyl
acetate, butanol and water. Alkaloids, tannins, resins, steroids, phenols and carbohydrate were found to be the
secondary metabolites present in P. pellucida. Brine shrimp lethality tests revealed that the methanol (LC50
260.89 μg/ml), hexane (LC50 333.91 μg/ml) and ethyl acetate (LC50 45.85 μg/ml) fractions were toxic while the
most polar fractions - butanol and aqueous fractions were non-toxic. LC50 ≥1000 μg/ml is considered non-toxic.
P. pellucida plant was also found to possess broad spectrum antimicrobial activity against Escherichia coli,
Staphylococcus aereus, Bacillus subtilis, Pseudomonas aeruginosa, Klebsiellae pneumonae, Salmonellae typhi,
Candida albicanas, Rhizopus stolon, Aspergillus niger and Penicillum notatum. The antioxidant activity of P.
pellucida as determined by three methods namely: scavenging effect on 2,2-diphenyl-1-picryhydrazyl radical
(DPPH), hydroxyl radical and ferric thiocyanate method, revealed that the fractions possessed antioxidant
activity when compared with antioxidant standards: butylated hydroxyl anisole (BHA), ascorbic acid and α-
tocopherol used in the assay. The extracts were however more active in the Ferric thiocyanate method giving a
% inhibition of over 98% scavenging activity. The high antioxidant activity of the plant at low concentration
indicates that it could be very useful for the treatment of ailments resulting from oxidative stress. These results
further corroborate the ethno medicinal uses of the plant.

Key words: Toxicity, antimicrobial, antioxidant, Peperomia pellucida, 2, 2-diphenyl-1-picrylhydrazyl radical,

hydroxyl radical, ferric thiocyanate.

Introduction maintain complex systems of multiple types of

antioxidants, such as glutathione, vitamin C and
Plants are free gifts of nature available at human vitamin E as well as enzymes such as catalase,
disposal for various pharmacological uses. They are superoxide dismutase and various peroxidases.
therefore, the source of active chemical compounds Oxidation reactions can produce excess free radicals
such as alkaloids tannins, flavonoids, sugars phenols, which start chain reactions that damage cells.
coumarins, terpenoids, saponins, etc useful to both Antioxidants terminate these chain reactions by
Man and animals. The rise in resistance of the human removing free radical intermediates and inhibit other
body system to certain orthodox medicine has called oxidation reactions. In recent times, antioxidant
for urgent exploration of natural products for chemistry has gained a lot of attention due to threats
medicinal uses. Research carried out on medicinal to biological macromolecules by oxidative processes.
plants is directed towards the isolation of active Plants are the major focus of researchers for isolation
compounds and once isolated, the active components of antioxidant secondary metabolites [21,8,38,25].
found in these plants are processed for the purpose of Peperomia pellucida also called Silver bush
administering them in a sufficiently and more precise belongs to the family Piperaceae. It is a herbaceous
dosage. The reliance of the pharmaceutical industry plant found in many South American and Asian
on organic Chemistry is therefore immense countries. The plant species has a history of ethno-
[10,11,9,39,42,24,2]. medicinal uses which include treatment of abdominal
Although oxidation reactions are crucial for life, pain, abscesses, acne, boils, colic, fatigue, gout
they can also be damaging hence, plants and animals headache, renal disorders, rheumatic pain and to treat
Corresponding Author
Ganiyat Kehinde Oloyede (Ph.D), N a t u r a l p r o d u c t s / M e d i c i n a l Chemistry Unit,
D e p a r t m e n t o f C h e m i s t r y , U n i v e r s i t y o f I b a d a n , N i g e r ia .
E-mail: oloyedegk @ gmail.com Telephone: +234 803 562 2238
 

Adv. Environ. Biol., 5(12): 3700-3709, 2011
breast cancer, impotence, measles, mental disorders
and small pox. However, there has not yet been
validated clinical data with regards to Peperomia
pellucida dosing. The plant’s contraindications
suggest that patients with known hypersensitivity
reactions to any of the components of the plant
species should avoid its use. The plant species also
interferes with prostaglandin synthesis which is
contraindicating for nursing mothers. Other
medicinal properties vary depending on region. In
Guyana, the plant has been used to lower cholesterol
and has been used as a cough suppressant, diuretic,
emollient and treatment of cardiac arrhythmia in the
Amazon region [7,40,3,10,32]. Numerous chemical
investigations, primarily on the essential oils of the
plant are found in medical literature. One study
identified 71 compounds from the essential oils of 10
piperaceae species. Flavonoids and phytosterols such
as acacetin, apigenin, isovitexin, pellucidatin
campesterol and stigmasterol, substituted styrenes
and pellucidin A have also been documented. Other
compounds like the peperomins with in vitro
cytotoxic or anticancer activity and arylpropanoids
such as the apiols with antifungal activity have been
isolated from Piperomia species [29,43,37,6,
44,14,4].
This research work is aimed at determining the
toxicity of the plant (using Brine shrimp larvae eggs),
screening the different extracts for antimicrobial
(using agar well diffusion technique) and antioxidant
activities. The antioxidant property was determined
by three methods not yet reported in literature for this
plant namely: scavenging effect on 2,2-Diphenyl-1-
picrylhydrazyl radical (DPPH), hydroxyl radical
generated by hydrogen peroxide and peroxide
oxidation by ferric thiocyanate method. Butylated
hydroxyanisole (BHA), ascorbic acid and -
tocopherol were used as reference standards [41].
The mechanism of action of the antioxidant effect of
P. Pellucida was also determined in this assay
[21,8,38,25].
Materials And Methods
Chemicals and Reagents:
Hexane, ethyl acetate, methanol, butanol,
chloroform, hydrochloric acid, ammonia solution,
naphthol, bismuth nitrate, potassium iodide, sodium
hydroxide, copper acetate, NaOH, sodium chloride,
copper sulphate pentahydrate, ferric chloride, conc.
tetraoxosulphate (VI) acid, Conc. HCl, ammonia
solution, sodium potassium tartarate, linoleic acid,
ammonium thiocyanate, ethanol, ferrous chloride,
hydrochloric acid, potassium chloride, glacial acetic
acid, disodium hydrogen phosphate, and dihydrogen
potassium phosphate were all BDH general purpose
chemicals and distilled prior to use.
Dimethylsulphoxide (M&B, England), hydrogen
peroxide (Merck, Germany) and 2, 2 - diphenyl-1-
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picrylhydrazyl (DPPH), ascorbic acid, Butylated
hydroxylanisole (BHA) and α-tocopherol were
obtained from Sigma Chemical Co (St Louis, MO).
Brine shrimp larvae eggs were obtained from Ocean
Star International, Inc. Company, USA.
Equipment and Apparatus:
Soxhlet apparatus, Mettler analytical balance
H80 (UK), Water Bath (Gallenkamp), Rotavapor
RII0 (Buchi, England), silica gel GF254 (precoated
aluminium sheets - Merck Germany), pH meter
(Jenway model), UV-Visible spectrophotometer
(UVD-2960 model equipped with a UVWIN
software version LABOMED INC, USA).
Plant collection and identification:
P. pellucida was identifified by Dr. L.S. Adebisi,
Head of the Department of Forestry and Wildlife of
the Faculty of Agriculture University of Ibadan and
were collected from the vicinity of Botany and
Microbiology Department, University of Ibadan, Oyo
State in June, 2010. Fresh plant (12.45kg) collected
was air dried to a constant weight (1.35kg) after 23
days.
Test Organisms:
Escherichia coli, Staphylococcus aereus,
Bacillus subtilis, Pseudomonas aeruginosa,
Klebsiellae pneumonae, Salmonellae typhi, Candida
albicans, Rhizopus stolon,, Aspergillus niger and
Penicillum notatum (Micro organisms were collected
from the stock of the Dept of Pharmaceutical
Microbiology, Faculty of Pharmacy of University of
Ibadan). The test organisms were maintained on
nutrient agar slopes and kept in a refrigerator at 4 oC.
100 ml aliquots of nutrient broth were inoculated
with the culture of test micro-organisms using a loop
and then incubated at 37 oC for 24 hrs.
Reference Standards:
Gentamicin at 10 mg/ml for bacteria and
Tioconazole (70%) for fungi both for antimicrobial
activity; Ascorbic acid, Butylated hydroxyanisole
(BHA) and α-Tocopherol for antioxidant activity.
Sample preparation:
Peperomia pellucida was collected, weighed and
air-dried for 23 days to a constant weight and then
pulverized using mill machine. The pulverized
samples were weighed and kept for further analysis.
Extraction/ partitioning procedure:
Solvent extraction of the plant (1.35 kg) was
carried out using 2.5 l of methanol in a soxhlet

Adv. Environ. Biol., 5(12): 3700-3709, 2011
apparatus. The extracts were collected, concentrated
and stored in a desiccator prior to analysis. Thin
Layer Chromatography (TLC) was employed using
silica gel 60 F254 precoated plates and solvent system:
Ethyl acetate/methanol (8:2) to detect antioxidant
activity with 2, 2-diphenyl-1-picrylhydrazylradical
(DPPH) as a spray reagent. Yellow coloration on the
spots on the TLC plates indicated that the methanolic
extract of P. pellucida had antioxidant activity. The
crude methanolic extract was then partitioned
successively in hexane, ethyl acetate, butanol and
water. Thereafter, toxicity test using Brine shrimp
lethality assay, antimicrobial screening by agar well
diffusion method and free radical scavenging activity
tests were carried out on the fractions using the
following spectrophotometric experiments:
scavenging effect on DPPH, scavenging effect on
hydroxyl radical generated by hydrogen peroxide and
peroxide oxidation by ferric thiocyanate method.
Phytochemical screening:
Tests for the presence of the following plant
secondary metabolites: alkaloids, flavonoids,
steroids, saponins, phenols, tannins, glycosides,
reducing sugars, anthraquinones, carbohydrates, resin
and cardiac glycosides were carried out on the crude
methanolic extract [22].
Toxicity analysis:
Brine shrimp lethality test:
The brine shrimp lethality test (BST) was used
to determine the toxicity of the fractions [30]. The
shrimp’s eggs were hatched in sea water for 48 h at
room temperature. The nauplii (harvested shrimps)
were attracted to one side of the vials with a light
source. Solutions of the extracts were made in
DMSO, at varying concentrations (1000, 100, and 10
µg/ml) and incubated in triplicate vials with the brine
shrimp larvae. Ten brine shrimp larvae were placed
in each of the triplicate vials. Control brine shrimp
larvae were placed in a mixture of sea water and
DMSO only. After 24 h the vials were examined
against a lighted background and the average number
of larvae that survived in each vial was determined.
The concentration at fifty percent mortality of the
larvae (LC50) was determined using the Finney
computer programme [17,35].
Antimicrobial screening methods:
Preparation of samples for Antimicrobial analysis:
The samples used for this study were: crude
methanolic extract, n-hexane, ethyl acetate and
butanol fractions of the plant. One gram of each
sample was weighed and dissolved in 5mls of the
solvent used for the extraction to give 200 mg/ml.
3702
This was serially diluted until a concentration of 6.25
mg/ml of the content was obtained in the sixth test
tube. The seventh test tube contained the solvent of
dissolution only (negative control). The eighth test
tube served as the positive control and contained
gentamicin for bacteria, tioconazole for fungi.
Agar diffusion: Pour plate method for bacterial:
An overnight culture of each organism
Staphylococcus aureus, Escherichia coli, Bacillus
subtilis, Pseudomonas aeruginosa, Klebsiellae
pneumonae and Salmonellae typhi was prepared. 0.1
ml of each of the organism was taken into 9.9 ml of
Sterile Distilled Water (SDW) to give 10 ml of 1:
100 (102) dilution. 0.2 ml was taken into the prepared
molten Nutrient Agar (NA) at 45 0C and this was
aseptically poured into the sterile plates and allowed
to set on the bench for 45 minutes. The stock was
maintained on nutrient agar slant and sub-cultured in
nutrient broth for incubation at 37 °C prior to each
antimicrobial testing. Inoculation of the test
organisms on nutrient agar-prepared plates was
achieved by flaming a wire loop on a spirit lamp,
cooling the wire loop (air cooling) and fetching the
test organisms. The discs were prepared using a
Grade No. 1 Whatman filter paper. 100 discs were
obtained by punching and putting in vials-bottles and
sterilizing in an oven at 150 °C for 15 min.
Thereafter the cups (9mm diameter) were aseptically
bored into the solid nutrient agar using a sterile cork
borer. A sterile cork-borer was used to create wells
(or holes) inside the set plates. The test solutions of
oils (50 μl) at concentration of 40 mg/ml were then
introduced into each of the designated cups on each
plate ensuring that no spillage occurred. The same
amount of the standard antimicrobial agent and
solvents were introduced were introduced using
syringes into the remaining cups on each plate to act
as positive and negative controls respectively. The
plates were left at room temperature for 2 hours,
allowed to diffuse into the medium, turned upside-
down and thereafter incubated at 37 0C for 24 hrs in
an incubator. Clear zones of inhibition were
observed. Activity or inactivity of each extract was
tested in triplicate and the diameters of zones of
inhibition were measured in millimetre (mm) using a
transparent well-calibrated ruler. The positive control
for bacteria is gentamicin at the concentration of 10
mg/ml. The analysis was done in triplicates and the
average readings were calculated [12,26,13].
Agar diffusion: Pour plate method for fungi:
Molten sterile Sabouraud Dextrose Agar (SDA)
was poured aseptically into the sterile plates and
allowed to cool down for 45 minutes. 0.2 ml of 1:100
dilution of the organisms Candida albicans,
Rhizopus stolon,, Aspergillus niger and Penicillum
notatum were spread on the surface using a sterile
spreader. Then, a sterile cork-borer was used to

Adv. Environ. Biol., 5(12): 3700-3709, 2011
create wells inside the plates. The same procedure
described for anti-bacterial activity above was
followed from this stage. The positive control for the
fungi is 70% tioconazole. All the plates for the fungi
were incubated at 28 0C for 48 hours unlike that of
bacteria that was incubated at 37 0C for 24 hours.
The clear zones of inhibition were observed and
recorded using the same method as described in the
case of bacteria [20,5].
Antioxidant activities of P. pellucida extracts:
Scavenging Effect on DPPH:
The antioxidant activity or the capacity to
scavenge the “stable” free radical DPPH was
determined using the DPPH free – radical scavenging
method. A 3.94 mg of 2, 2-diphenyl-1-picryhydrazyl
radical (DPPH), a stable radical was dissolved in
methanol (100ml) to give a 100 µm solution. To 3.0
ml of the methanolic solutions of DPPH was added
0.5 ml of each of the fractions with doses ranging
from 1.0 mg/ml to 0.0625 mg/ml [19,31,35]. The
decrease in absorption at 517 nm of DPPH was
measured 10 minutes later. The actual decrease in
absorption was measured against that of the control
and the percentage inhibition was also calculated.
The same experiment was carried out on butylated
hydroxylanisole (BHA), α-tocopherol and ascorbic
acid which are known antioxidants. All test and
analysis were run in triplicates and the results
obtained were averaged. The radical scavenging
activity (RSA) was calculated as the percentage
inhibition of DPPH discoloration using the equation
below:
%RSA or % inhibition = {(ADPPH – AS)/ADPPH} x 100
Where AS is the absorbance of the solution and
ADPPH is the absorbance of the DPPH solution [23].
Scavenging Effect on Hydrogen Peroxide:
Spectrophotometric determination of the
fractions of P. pellucida was carried out at 285 nm.
A solution of 2 mM hydrogen peroxide was prepared
in phosphate buffered-saline (PBS) pH 7.4. The
fractions at the following concentrations; 0.1-
0.00625 mg/ml was added to the H2O2 solution.
Decrease in absorbance of H2O2 at 285nm was
determined spectrophotometrically 10 minutes later
against a blank solution containing the test extract in
PBS without H2O2. All tests were run in triplicates
and averaged [41,36]. The same experiment was
carried out on Butylatedhydroxyanisole (BHA),
ascorbic acid and α-tocopherol which are known
antioxidant standards.
Antioxidant activity by ferric thiocyanate method:
The antioxidant activities of hexane, ethyl
acetate and butanol fractions of the plant material
3703
were determined by ferric thiocyanate method [28].
10 mg of each extract was dissolved separately in
99.5% of ethanol and various concentrations (50,
100, 250, 500 μg/ml) were prepared. A mixture of a
2 ml of sample in 99.5% ethanol, 2.0 ml of 2.51%
linoleic acid in 99.5% ethanol, 4 ml of 0.05 M
phosphate buffer (pH 7.0) and 2 ml of water was
placed in a vial with a screw cap and placed in an
oven at 600C in the dark. To 0.1 ml of this sample
solution, 10 ml of 75% ethanol and 0.1 ml of 30%
ammonium thiocynate was added. After the addition
of 0.1 ml of 2 x 10-2 M ferrous chloride in 3.5%
hydrochloric acid to the reaction mixture, the
absorbance of the red colour developed was
measured in 3 min at 500 nm. The control and
standards were subjected to the same procedures as
the sample, except that for the control, only solvent
was added, and for the standard, sample was replaced
with the same amount of Butylatedhydroxyanisole
(BHA), ascorbic acid and α-tocopherol (reference
compounds)[35]. The inhibition of lipid peroxidation
in percentage was calculated using this equation:
% Inhibition = 1 - (A1/A2) X 100
Where A1 was the absorbance of the test sample and
A2 was the absorbance of control reaction.
Results And Discussion
The percentage moisture content of the leaves of
Peperomia pellucida was 89.16%. The crude
methanol extract of P. pellucida was found to contain
alkaloids, tannins, resins, flavonoids, steroids,
phenols and carbohydrates as reported by other
workers.
Brine shrimp lethality test:
The toxicity result of the extracts obtained from
of P. pellucida showed that the fractions (methanol,
hexane and ethylacetate) are toxic to brine shrimp
larvae at different lethal concentrations (LC50) while
the butanol and aqueous fractions were not toxic
having LC50 values greater than 1000 μg/ml. Toxicity
level as determined by Finney computer programme
gave the following lethal concentration, Methanol
fraction, LC50 = 260.89 μg/ml, hexane fraction, LC50
= 333.91 μg/ml, ethyl acetate fraction, LC50= 45.85
μg/ml, butanol fraction, LC50 = 2105.63 μg/ml and
aqueous fraction 3152.61 μg/ml (Table 1). The result
corroborated the presence in the plant of medicinally
active compounds. The use of Peperomia pellucida
is reported to be contraindicating in lactation as it is
also said to interfere with prostaglandin synthesis [1].
The toxicity result obtained in our assay also buttress
this observation. However the ingestion of the polar
fractions (aqueous and butanol) of Peperomia
pellucida may not be harmful to the human body due
to its non-toxicity.
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Adv. Environ. Biol., C(): CC-CC, 2011

Table 1: Results of Brine shrimp lethality test of Peperomia pellucida leaf extracts*
Conc./ 10000ppm 1000ppm 100ppm Control
Sample S D S D S D S D LD50 µg/ml
CPP 0 30 12 18 18 12 10 0 260.8919
HFPP 3 27 9 21 21 9 10 0 333.9137
EAFPP 0 30 5 25 11 19 10 0 45.8487
BFPP 8 22 19 11 26 4 10 0 2105.6330
AFPP 10 20 19 11 29 1 10 0 3152.6070
*LD50 < 1000 µg/ml =Toxic, LD50 > 1000 µg/ml = Not Toxic S- Survivor, D-Death, CPP- Crude extract of P. pellucida, HFPP- Hexane
Fraction of P. pellucida, EAFPP- Ethyl Acetate Fraction of P. pellucida, BFPP- Butanol Fraction of P. pellucida, AFPP
Aqueous Fraction of P. pellucida

Cytotoxicity results of Peperomia pellucida leaf
extracts:
Antimicrobial Screening:
P. pellucida extracts were screened at various
concentrations for antimicrobial activity using the
Agar well diffusion method. The zones of inhibition
(mm) were measured in triplicate and the average
results obtained is shown in Table 2-6. It was
observed that all the tested samples possessed
antimicrobial activities indicating a broad spectrum
on both gram positive and gram negative bacteria
and the fungi used.

Table 2: Antimicrobial activity of crude methanol fraction of P. pellucida leaves

Zones of Inhibition (mm)
Concentration S.a E.coli B.sub Ps.a Kleb Sal. C.a A.n Rhiz Pen
1 10 10 10 12 10 10 16 14 18 12
2 - - - 10 - - 4 12 16 10
3 - - - - - - 12 10 12 -
4 - - - - - - 10 - 10 -
5 - - - - - - - - - -
6 - - - - - - - - - -
-ve - - - - - - - - - -
+ve 38 34 36 36 34 30 26 24 22 24
*Integers 1-6 represent methanol fraction at various concentrations viz: (1) 200mg/ml, (2) 100mg/ml, (3) 50mg/ml, (4) 25mg/ml, (5)
12.5mg/ml, (6) 6.25mg/ml -ve= negative control (methanol), +ve= positive control {Gentamicin at 10 mg/ml for bacteria or Tioconazole
(70%) for fungi} - = no inhibition, S.a= Staphylococcus aureus, E. coli= Escherichia coli, B. Sub= Bacillus subtilis, Ps.a= Pseudomonas
aeruginosa, Kleb= Klebsiellae pneumonae, Sal= Salmonellae typhi, C.a= Candida albicanas, A.n= Aspergillus niger, Rhiz = Rhizopus
stolon, Pen= Penicillum notatum.

Table 3: Antimicrobial activity of n-hexane fraction of P. pellucida leaves.

Zones of Inhibition (mm)
Concentration S.a E.coli B.sub Ps.a Kleb Sal. C.a A.n Rhiz Pen
1 16 16 16 18 16 18 20 18 14 12
2 14 14 14 16 14 16 16 16 12 10
3 12 12 12 14 12 14 12 12 10 -
4 10 10 10 12 10 12 10 10 - -
5 - - - 10 - 10 - - - -
6 - - - - - - - - - -
-ve - - - - - - - - - -
+ve 38 36 34 36 34 36 24 26 24 26
*Integers 1-6 represent hexane fraction at various concentrations viz: (1) 200mg/ml, (2) 100mg/ml, (3) 50mg/ml, (4) 25mg/ml, (5)
12.5mg/ml, (6) 6.25mg/ml -ve= negative control (hexane), +ve= positive control {Gentamicin at 10 mg/ml for bacteria or Tioconazole
(70%) for fungi} - = no inhibition, S.a= Staphylococcus aureus, E. coli= Escherichia coli, B. Sub= Bacillus subtilis, Ps.a= Pseudomonas
aeruginosa, Kleb= Klebsiellae pneumonae, Sal= Salmonellae typhi, C.a= Candida albicanas, A.n= Aspergillus niger, Rhiz = Rhizopus
stolon, Pen= Penicillum notatum.

Table 4: Antimicrobial activity of ethyl acetate fraction of P. pellucida leaves.

Zones of Inhibition (mm)
Concentration S.a E.coli B.sub Ps.a Kleb Sal. C.a A.n Rhis Pen
1 14 10 16 14 18 14 14 16 18 20
2 12 - 14 10 16 12 10 14 14 16
3 10 - 12 - 12 10 - 12 12 12
4 - - 10 - 10 - - 10 10 10
5 - - - - - - - - - -
6 - - - - - - - - - -
-ve - - - - - - - - - -
+ve 38 36 34 36 34 36 24 26 24 26
*Integers 1-6 represent ethylacetate fraction at various concentrations viz: (1) 200mg/ml, (2) 100mg/ml, (3) 50mg/ml, (4) 25mg/ml, (5)
12.5mg/ml, (6) 6.25mg/ml -ve= negative control (ethylacetate), +ve= positive control {Gentamicin at 10 mg/ml for bacteria or Tioconazole
(70%) for fungi} - = no inhibition, S.a= Staphylococcus aureus, E. coli= Escherichia coli, B. Sub= Bacillus subtilis, Ps.a= Pseudomonas
aeruginosa, Kleb= Klebsiellae pneumonae, Sal= Salmonellae typhi, C.a= Candida albicanas, A.n= Aspergillus niger, Rhiz – Rhizopus
stolon, Pen= Penicillum notatum.

 
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Adv. Environ. Biol., C(): CC-CC, 2011

Table 5: Antimicrobial activity of butanol fraction of P. pellucida leaves.

Zones of Inhibition (mm)
Concentration S.a E.coli B.sub Ps.a Kleb Sal. C.a A.n Rhis Pen
1 22 12 12 20 14 16 20 14 20 18
2 18 10 10 16 12 14 16 12 16 14
3 16 - - 14 10 10 14 10 12 12
4 14 - - 12 - - 10 - 10 10
5 10 - - - - - - - - -
6 - - - - - - - - - -
-ve - - - - - - - - - -
+ve 38 36 34 36 34 36 24 26 24 26
*Integers 1-6 represent butanol fraction at various concentrations viz: (1) 200mg/ml, (2) 100mg/ml, (3) 50mg/ml, (4) 25mg/ml, (5)
12.5mg/ml, (6) 6.25mg/ml -ve= negative control (butanol), +ve = positive control {Gentamicin at 10 mg/ml for bacteria or Tioconazole
(70%) for fungi} - = no inhibition, S.a= Staphylococcus aureus, E. coli= Escherichia coli, B. Sub= Bacillus subtilis, Ps.a= Pseudomonas
aeruginosa, Kleb= Klebsiellae pneumonae, Sal= Salmonellae typhi, C.a= Candida albicanas, A.n= Aspergillus niger, Rhiz = Rhizopus
stolon, Pen= Penicillum notatum.

Table 6: Antimicrobial activity of aqueous fraction of P. pellucida leaves.

Zones of Inhibition (mm)
Concentration S.a E.coli B.sub Ps.a Kleb Sal. C.a A.n Rhiz Pen
1 14 12 10 18 20 14 14 16 12 12
2 12 10 - 16 16 12 12 14 10 10
3 10 - - 14 12 10 10 12 - -
4 - - - 12 10 - - 10 - -
5 - - - - - - - - - -
6 - - - - - - - - - -
-ve - - - - - - - - - -
+ve 36 34 36 34 36 36 24 26 24 26
*Integers 1-6 represent aqueous fraction at various concentrations viz: (1) 200mg/ml, (2) 100mg/ml, (3) 50mg/ml, (4) 25mg/ml, (5)
12.5mg/ml, (6) 6.25mg/ml -ve= negative control (water), +ve = positive control {Gentamicin at 10 mg/ml for bacteria or Tioconazole
(70%) for fungi} - = no inhibition, S.a= Staphylococcus aureus, E. coli= Escherichia coli, B. Sub= Bacillus subtilis, Ps.a= Pseudomonas
aeruginosa, Kleb= Klebsiellae pneumonae, Sal= Salmonellae typhi, C.a= Candida albicanas, A.n= Aspergillus niger, Rhiz = Rhizopus
stolon, Pen= Penicillum notatum.

The different fractions examined inhibited the Antioxidant Activity:

micro-organism Escherichia coli, Staphylococcus
aereus, Bacillus subtilis, Pseudomonas aeruginosa,
Klebsiellae pneumonae, Salmonellae typhi, Candida
albicans, Rhizopus stolon, Aspergillus niger and
Penicillum notatum in a concentration dependent
manner. Activity was generally observed at the
higher concentrations 25-200 mg/ml. The activity of
the extracts though high were not as active as the
reference compounds, the positive control
(gentamicin at 10mg/ml for bacteria and tioconazole
(70 %) for fungi. The methanolic extract was active
on C. albicans and R. stolon at 25-200 mg/ml which
indicates a selective inhibition on fungi (Table 2).
The n–hexane fraction (Table 3) inhibited all the
microbes at 50 - 200 mg/ml but showed mild activity
on R. stolon and P. notatum. The ethyl acetate
fraction exhibited a broad spectrum activity on the
entire microorganisms at 50 - 200 mg/ml except on
E.coli (Table 4). Table 5 showed the activity of the
butanol fraction on tested microbes. It showed
moderate activity for all but was particularly active
on S. aureus at 12.5-200 mg/ml. The aqueous
fraction (Table 6) inhibited P. aeruginosa, K.
pneumonae, S. typhi, C. albicans and A. niger
moderately at 50-200 mg/ml. Overall, it was
observed that the crude methanolic extract was low
in activity compared to the other fractions while the
hexane, ethyl acetate and butanol fractions at
concentration of 50 - 200 mg/ml showed a broad
spectrum antimicrobial activity on bacteria and the
fungi used in this assay.
The antioxidant activities of the methanol,
hexane, ethyl acetate, butanol and aqueous extracts
of P. pellucida were determined by three methods:
scavenging effect on 2, 2-diphenyl-1-picryhydrazyl
radical (DPPH), hydroxyl radical generated by
hydrogen peroxide and ferric thiocyanate (FTC)
method. The results are presented in Tables 7-10.
Scavenging effects on DPPH:
The reduction in absorbance of DPPH at 517nm
caused by the samples was measured in triplicate
after 10min. The tested samples showed very good
activity when compared to the standard used (Table
5). There was decrease in absorption at 517 nm
indicating that the fractions have hydrogen donating
ability or can scavenge free radical. The observation
was further corroborated by the calculated
percentage inhibition. Fractions from P. pellucida
have good activities as free radical scavengers when
compared with controls: ascorbic acid, Butylated
hydroxylanisole (BHA) and α –Tocopherol (Table
7). All the fractions were moderately active having
%inhibition of 51 - 99% at 1.0 – 0.0625 mg/ml.
Butanol has the highest scavenging activity with %
inhibition of 98.6% at concentration of 0.0625 mg/ml
(Table 8). Generally, all the fractions tested have
better antioxidant activity than the reference
standards, ascorbic acid, BHA and α –tocopherol and
inhibition increased with decrease in concentration.

 
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Table 7: Absorbance values from scavenging effect of fractions from P. pellucida on DPPH at 517 (nm)* .
CONC. P1 P2 P3 P4 P5 ASCORBIC BHA α-
( mg/ml) ACID TOCOPHEROL
1.0 0.0907±0.006 0.2138±0.002 0.4525±0.001 0.1261±0.001 0.2019±0.002 0.0843±0.010 0.0370±0.006 0.6800±0.029
0.5 0.1049±0.001 0.1290±0.005 0.2436±0.002 0.1137±0.001 0.1340±0.001 0.2893±0.128 0.0460±0.008 0.7040±0.003
0.25 0.1148±0.001 0.1108±0.001 0.1851±0.003 0.0912±0.003 0.1109±0.003 0.2977±0.124 0.0483±0.002 0.7047±0.007
0.125 0.0836±0.002 0.1059±0.001 0.1368±0.003 0.0842±0.001 0.1028±0.002 0.3200±0.082 0.0490±0.004 0.7070±0.007
0.0625 0.0868±0.001 0.0843±0.003 0.1120±0.001 0.1061±0.001 0.1009±0.003 0.5147±0.015 0.0650±0.003 0.7207±0.012
*Absorbance measurement of P1 = crude methanol extract, P2 = n-Hexane fraction, P3 = Ethyl acetate fraction, P4 = Butanol fraction, P5 = Aqueous fraction, Ascorbic Acid, BHA and α-
Tocopherol at 517nm.

Table 8: Percentage inhibitions of fractions from P. pellucida and standards*.

Conc.(mg/ml) P1 P2 P3 P4 P5
Ascorbic BHA α-
acid Tocopherol
1.0 89.6 77.2 51.4 86.5 78.4 90.9 95.4 15.4
0.5 88.6 85.1 73.7 87.9 85.8 68.7 94.3 12.4
0.25 87.6 85.5 79.8 90.3 87.7 67.8 94.00 12.4
0.125 90.7 88.7 85.0 90.9 88.9 65.4 93.9 12.1
0.0625 90.8 91.0 87.8 98.6 88.9 44.3 91.9 10.4
*Percentage inhibition of the plant P. pellucida and the standard using the formula % inhibition = ADPPH – As / ADPPH x 100

Scavenging effects on Hydrogen peroxide (H2O2): the n-hexane fraction. All the fractions showed better
activity than the standards. P. pellucida therefore is a
Scavenging effects on H2O2 was measured in source of antioxidant compounds especially in
triplicates after 10min of incubation at 285nm. The scavenging the highly reactive hydroxyl radicals
scavenging activities of fractions and antioxidants which are known to cause oxidative damage to
like ascorbic acid, Butylated hydroxyanisole (BHA) biological macromolecules.
and α-tocopherol on H2O2 is shown in Table 9. It has
been observed that H2O2 through the Fenton reaction Antioxidant activity by Ferric thiocyanate method
is an active - oxygen specie and has potential to (FTC):
produce the highly reactive hydroxyl radical which
are often involved in free radical chain reactions In this method, the concentration of peroxide
(Namiki, 1990 and Lugasi et al., 1999). decreased as the antioxidant activity increased (Table
Hydroxyl radical scavenging ability of the 10). The FTC method was used to determine the
fractions from P. pellucida is seen in Table 9 and amount of peroxide which oxidized ferrous chloride
Figure 1. At concentration of 0.1 - 0.0065 mg/ml, the (FeCl2) to a reddish ferric chloride (FeCl3) pigment.
fractions had high scavenging activities when Hexane, ethyl acetate, butanol, crude methanol and
compared to standards. The % inhibition was aqueous extracts at various concentration (0.00625 –
between 92-99% at all the concentrations used (Fig 0.8 mg/ml), showed moderate antioxidant activities
1). Activity was found to be better particularly with in a concentration dependent manner.
Table 9: Scavenging Effect of H2O2 on the fractions from P. pellucida Extract*.
CONC (mg/ml) P1 P2 P3 P4 P5 ASCORBIC ACID BHA ALPHA
TOCOPHEROL
0.1 0.057±0.000 0.195±0.001 0.704±0.001 0.272±0.000 0.220±0.040 0.1952±0.001 0.0413±0.016 0.0321±0.045
0.05 0.056±0.000 0.134±0.002 0.320±0.000 0.184±0.001 0.114±0.000 0.2078±0.012 0.0617±0.019 0.0633±0.032
0.025 0.048±0.000 0.085±0.001 0.179±0.000 0.109±0.000 0.083±0.001 1.2645±0.119 0.0740±0.015 0.1552±0.061
0.0125 0.265±0.001 0.075±0.000 0.116±0.001 0.119±0.000 0.066±0.000 2.7586±0.049 0.0947±0.003 0.1807±0.015
0.00625 0.052±0.000 0.113±0.002 0.112±0.001 0.065±0.002 0.057±0.002 2.9236±0.211 0.1126±0.014 0.4940±0.017
*Absorbance measurement of P1 = n-Hexane fraction, P2 = Ethyl acetate fraction, P3 = Butanol fraction, P4 = crude methanol extract, P5 = Aqueous fraction, Ascorbic Acid, BHA and α-
Tocopherol at 285nm

Fig. 1: H2O2 Free radical scavenging activity of the extracts from the plant of P. pellucida and standards at 285
nm measured in triplicate. P1 = n-Hexane fraction, P2 = Ethyl acetate fraction, P3 = Butanol fraction, P4
= crude methanol extract, P5 = Aqueous fraction.

 
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Table 10: Peroxide oxidation of fractions from P. pellucida Extract at 500 nm using the Ferric thiocyanate method*.
CONC (mg/ml) P1 P2 P3 P4 P5 ASCORBIC BHA ALPHA
ACID TOCOPHEROL
0.8 0.478±0.005 0.420±0.023 0.495±0.085 0.341±0.044 0.297±0.008 0.173±0.008 0.326±0.006 0.133±0.004
0.4 0.182±0.006 0.702±0.020 0.425±0.016 0.485±0.011 0.423±0.064 0.173±0.008 0.375±0.008 0.164±0.006
0.2 0.289±0.014 0.318±0.003 0.472±0.005 0.216±0.009 0.179±0.015 0.245±0.008 0.431±0.008 0.184±0.009
0.1 0.381±0.004 0.244±0.006 0.306±0.003 0.054±0.001 0.127±0.003 0.275±0.006 0.616±0.005 0.195±0.023
0.05 0.476±0.020 0.134±0.007 0.644±0.013 0.162±0.001 0.151±0.001 0.287±0.050 0.647±0.004 0.294±0.004
0.025 0.216±0.002 0.120±0.003 0.335±0.006 0.224±0.027 0.188±0.002 0.367±0.004 0.653±0.008 0.340±0.069
0.0125 0.379±0.004 0.129±0.005 0.258±0.001 0.132±0.001 0.171±0.002 0.516±0.008 0.747±0.003 0.360±0.005
0.00625 0.576±0.001 0.126±0.000 0.359±0.011 0.105±0.001 0.129±0.001 0.668±0.002 0.750±0.001 0.377±0.008
*Absorbance measurement of P1 = n-Hexane fraction, P2 = Ethyl acetate fraction, P3 = Butanol fraction, P4 = crude methanol extract, P5 = Aqueous fraction, Ascorbic Acid, BHA and α-
Tocopherol at 500 nm

Fig. 2: Peroxide oxidation of the extracts from the whole plant of P. pellucida and standards at 500 nm
measured in triplicate. P1 = n-Hexane fraction, P2 = Ethyl acetate fraction, P3 = Butanol fraction, P4 =
crude methanol extract, P5 = Aqueous fraction.

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