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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
 
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Adv. Environ. Biol., 5(12): 3700-3709, 2011

breast cancer, impotence, measles, mental disorders picrylhydrazyl (DPPH), ascorbic acid, Butylated
and small pox. However, there has not yet been hydroxylanisole (BHA) and α-tocopherol were
validated clinical data with regards to Peperomia obtained from Sigma Chemical Co (St Louis, MO).
pellucida dosing. The plant’s contraindications Brine shrimp larvae eggs were obtained from Ocean
suggest that patients with known hypersensitivity Star International, Inc. Company, USA.
reactions to any of the components of the plant
species should avoid its use. The plant species also Equipment and Apparatus:
interferes with prostaglandin synthesis which is
contraindicating for nursing mothers. Other Soxhlet apparatus, Mettler analytical balance
medicinal properties vary depending on region. In H80 (UK), Water Bath (Gallenkamp), Rotavapor
Guyana, the plant has been used to lower cholesterol RII0 (Buchi, England), silica gel GF254 (precoated
and has been used as a cough suppressant, diuretic, aluminium sheets - Merck Germany), pH meter
emollient and treatment of cardiac arrhythmia in the (Jenway model), UV-Visible spectrophotometer
Amazon region [7,40,3,10,32]. Numerous chemical (UVD-2960 model equipped with a UVWIN
investigations, primarily on the essential oils of the software version LABOMED INC, USA).
plant are found in medical literature. One study
identified 71 compounds from the essential oils of 10 Plant collection and identification:
piperaceae species. Flavonoids and phytosterols such
as acacetin, apigenin, isovitexin, pellucidatin P. pellucida was identifified by Dr. L.S. Adebisi,
campesterol and stigmasterol, substituted styrenes Head of the Department of Forestry and Wildlife of
and pellucidin A have also been documented. Other the Faculty of Agriculture University of Ibadan and
compounds like the peperomins with in vitro were collected from the vicinity of Botany and
cytotoxic or anticancer activity and arylpropanoids Microbiology Department, University of Ibadan, Oyo
such as the apiols with antifungal activity have been State in June, 2010. Fresh plant (12.45kg) collected
isolated from Piperomia species [29,43,37,6, was air dried to a constant weight (1.35kg) after 23
44,14,4]. days.
This research work is aimed at determining the
toxicity of the plant (using Brine shrimp larvae eggs), Test Organisms:
screening the different extracts for antimicrobial
(using agar well diffusion technique) and antioxidant Escherichia coli, Staphylococcus aereus,
activities. The antioxidant property was determined Bacillus subtilis, Pseudomonas aeruginosa,
by three methods not yet reported in literature for this Klebsiellae pneumonae, Salmonellae typhi, Candida
plant namely: scavenging effect on 2,2-Diphenyl-1- albicans, Rhizopus stolon,, Aspergillus niger and
picrylhydrazyl radical (DPPH), hydroxyl radical Penicillum notatum (Micro organisms were collected
generated by hydrogen peroxide and peroxide from the stock of the Dept of Pharmaceutical
oxidation by ferric thiocyanate method. Butylated Microbiology, Faculty of Pharmacy of University of
hydroxyanisole (BHA), ascorbic acid and - Ibadan). The test organisms were maintained on
tocopherol were used as reference standards [41]. nutrient agar slopes and kept in a refrigerator at 4 oC.
The mechanism of action of the antioxidant effect of 100 ml aliquots of nutrient broth were inoculated
P. Pellucida was also determined in this assay with the culture of test micro-organisms using a loop
[21,8,38,25]. and then incubated at 37 oC for 24 hrs.

Materials And Methods Reference Standards:

Chemicals and Reagents: Gentamicin at 10 mg/ml for bacteria and

Tioconazole (70%) for fungi both for antimicrobial
Hexane, ethyl acetate, methanol, butanol, activity; Ascorbic acid, Butylated hydroxyanisole
chloroform, hydrochloric acid, ammonia solution, (BHA) and α-Tocopherol for antioxidant activity.
naphthol, bismuth nitrate, potassium iodide, sodium
hydroxide, copper acetate, NaOH, sodium chloride, Sample preparation:
copper sulphate pentahydrate, ferric chloride, conc.
tetraoxosulphate (VI) acid, Conc. HCl, ammonia Peperomia pellucida was collected, weighed and
solution, sodium potassium tartarate, linoleic acid, air-dried for 23 days to a constant weight and then
ammonium thiocyanate, ethanol, ferrous chloride, pulverized using mill machine. The pulverized
hydrochloric acid, potassium chloride, glacial acetic samples were weighed and kept for further analysis.
acid, disodium hydrogen phosphate, and dihydrogen
potassium phosphate were all BDH general purpose Extraction/ partitioning procedure:
chemicals and distilled prior to use.
Dimethylsulphoxide (M&B, England), hydrogen Solvent extraction of the plant (1.35 kg) was
peroxide (Merck, Germany) and 2, 2 - diphenyl-1- carried out using 2.5 l of methanol in a soxhlet

 
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apparatus. The extracts were collected, concentrated This was serially diluted until a concentration of 6.25
and stored in a desiccator prior to analysis. Thin mg/ml of the content was obtained in the sixth test
Layer Chromatography (TLC) was employed using tube. The seventh test tube contained the solvent of
silica gel 60 F254 precoated plates and solvent system: dissolution only (negative control). The eighth test
Ethyl acetate/methanol (8:2) to detect antioxidant tube served as the positive control and contained
activity with 2, 2-diphenyl-1-picrylhydrazylradical gentamicin for bacteria, tioconazole for fungi.
(DPPH) as a spray reagent. Yellow coloration on the
spots on the TLC plates indicated that the methanolic Agar diffusion: Pour plate method for bacterial:
extract of P. pellucida had antioxidant activity. The
crude methanolic extract was then partitioned An overnight culture of each organism
successively in hexane, ethyl acetate, butanol and Staphylococcus aureus, Escherichia coli, Bacillus
water. Thereafter, toxicity test using Brine shrimp subtilis, Pseudomonas aeruginosa, Klebsiellae
lethality assay, antimicrobial screening by agar well pneumonae and Salmonellae typhi was prepared. 0.1
diffusion method and free radical scavenging activity ml of each of the organism was taken into 9.9 ml of
tests were carried out on the fractions using the Sterile Distilled Water (SDW) to give 10 ml of 1:
following spectrophotometric experiments: 100 (102) dilution. 0.2 ml was taken into the prepared
scavenging effect on DPPH, scavenging effect on molten Nutrient Agar (NA) at 45 0C and this was
hydroxyl radical generated by hydrogen peroxide and aseptically poured into the sterile plates and allowed
peroxide oxidation by ferric thiocyanate method. to set on the bench for 45 minutes. The stock was
maintained on nutrient agar slant and sub-cultured in
Phytochemical screening: nutrient broth for incubation at 37 °C prior to each
antimicrobial testing. Inoculation of the test
Tests for the presence of the following plant organisms on nutrient agar-prepared plates was
secondary metabolites: alkaloids, flavonoids, achieved by flaming a wire loop on a spirit lamp,
steroids, saponins, phenols, tannins, glycosides, cooling the wire loop (air cooling) and fetching the
reducing sugars, anthraquinones, carbohydrates, resin test organisms. The discs were prepared using a
and cardiac glycosides were carried out on the crude Grade No. 1 Whatman filter paper. 100 discs were
methanolic extract [22]. 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
Toxicity analysis:
bored into the solid nutrient agar using a sterile cork
borer. A sterile cork-borer was used to create wells
Brine shrimp lethality test:
(or holes) inside the set plates. The test solutions of
oils (50 μl) at concentration of 40 mg/ml were then
The brine shrimp lethality test (BST) was used introduced into each of the designated cups on each
to determine the toxicity of the fractions [30]. The plate ensuring that no spillage occurred. The same
shrimp’s eggs were hatched in sea water for 48 h at amount of the standard antimicrobial agent and
room temperature. The nauplii (harvested shrimps) solvents were introduced were introduced using
were attracted to one side of the vials with a light syringes into the remaining cups on each plate to act
source. Solutions of the extracts were made in as positive and negative controls respectively. The
DMSO, at varying concentrations (1000, 100, and 10 plates were left at room temperature for 2 hours,
µg/ml) and incubated in triplicate vials with the brine allowed to diffuse into the medium, turned upside-
shrimp larvae. Ten brine shrimp larvae were placed down and thereafter incubated at 37 0C for 24 hrs in
in each of the triplicate vials. Control brine shrimp an incubator. Clear zones of inhibition were
larvae were placed in a mixture of sea water and observed. Activity or inactivity of each extract was
DMSO only. After 24 h the vials were examined tested in triplicate and the diameters of zones of
against a lighted background and the average number inhibition were measured in millimetre (mm) using a
of larvae that survived in each vial was determined. transparent well-calibrated ruler. The positive control
The concentration at fifty percent mortality of the for bacteria is gentamicin at the concentration of 10
larvae (LC50) was determined using the Finney mg/ml. The analysis was done in triplicates and the
computer programme [17,35]. average readings were calculated [12,26,13].

Antimicrobial screening methods: Agar diffusion: Pour plate method for fungi:

Preparation of samples for Antimicrobial analysis: Molten sterile Sabouraud Dextrose Agar (SDA)
was poured aseptically into the sterile plates and
The samples used for this study were: crude allowed to cool down for 45 minutes. 0.2 ml of 1:100
methanolic extract, n-hexane, ethyl acetate and dilution of the organisms Candida albicans,
butanol fractions of the plant. One gram of each Rhizopus stolon,, Aspergillus niger and Penicillum
sample was weighed and dissolved in 5mls of the notatum were spread on the surface using a sterile
solvent used for the extraction to give 200 mg/ml. spreader. Then, a sterile cork-borer was used to

 
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create wells inside the plates. The same procedure were determined by ferric thiocyanate method [28].
described for anti-bacterial activity above was 10 mg of each extract was dissolved separately in
followed from this stage. The positive control for the 99.5% of ethanol and various concentrations (50,
fungi is 70% tioconazole. All the plates for the fungi 100, 250, 500 μg/ml) were prepared. A mixture of a
were incubated at 28 0C for 48 hours unlike that of 2 ml of sample in 99.5% ethanol, 2.0 ml of 2.51%
bacteria that was incubated at 37 0C for 24 hours. linoleic acid in 99.5% ethanol, 4 ml of 0.05 M
The clear zones of inhibition were observed and phosphate buffer (pH 7.0) and 2 ml of water was
recorded using the same method as described in the placed in a vial with a screw cap and placed in an
case of bacteria [20,5]. 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%
Antioxidant activities of P. pellucida extracts: ammonium thiocynate was added. After the addition
of 0.1 ml of 2 x 10-2 M ferrous chloride in 3.5%
Scavenging Effect on DPPH: hydrochloric acid to the reaction mixture, the
absorbance of the red colour developed was
The antioxidant activity or the capacity to measured in 3 min at 500 nm. The control and
scavenge the “stable” free radical DPPH was standards were subjected to the same procedures as
determined using the DPPH free – radical scavenging the sample, except that for the control, only solvent
method. A 3.94 mg of 2, 2-diphenyl-1-picryhydrazyl was added, and for the standard, sample was replaced
radical (DPPH), a stable radical was dissolved in with the same amount of Butylatedhydroxyanisole
methanol (100ml) to give a 100 µm solution. To 3.0 (BHA), ascorbic acid and α-tocopherol (reference
ml of the methanolic solutions of DPPH was added compounds)[35]. The inhibition of lipid peroxidation
0.5 ml of each of the fractions with doses ranging
in percentage was calculated using this equation:
from 1.0 mg/ml to 0.0625 mg/ml [19,31,35]. The
% Inhibition = 1 - (A1/A2) X 100
decrease in absorption at 517 nm of DPPH was
Where A1 was the absorbance of the test sample and
measured 10 minutes later. The actual decrease in
A2 was the absorbance of control reaction.
absorption was measured against that of the control
and the percentage inhibition was also calculated.
The same experiment was carried out on butylated Results And Discussion
hydroxylanisole (BHA), α-tocopherol and ascorbic
acid which are known antioxidants. All test and The percentage moisture content of the leaves of
analysis were run in triplicates and the results Peperomia pellucida was 89.16%. The crude
obtained were averaged. The radical scavenging methanol extract of P. pellucida was found to contain
activity (RSA) was calculated as the percentage alkaloids, tannins, resins, flavonoids, steroids,
inhibition of DPPH discoloration using the equation phenols and carbohydrates as reported by other
below: workers.
%RSA or % inhibition = {(ADPPH – AS)/ADPPH} x 100
Where AS is the absorbance of the solution and Brine shrimp lethality test:
ADPPH is the absorbance of the DPPH solution [23].
The toxicity result of the extracts obtained from
Scavenging Effect on Hydrogen Peroxide: of P. pellucida showed that the fractions (methanol,
hexane and ethylacetate) are toxic to brine shrimp
Spectrophotometric determination of the larvae at different lethal concentrations (LC50) while
fractions of P. pellucida was carried out at 285 nm. the butanol and aqueous fractions were not toxic
A solution of 2 mM hydrogen peroxide was prepared having LC50 values greater than 1000 μg/ml. Toxicity
in phosphate buffered-saline (PBS) pH 7.4. The level as determined by Finney computer programme
fractions at the following concentrations; 0.1- gave the following lethal concentration, Methanol
0.00625 mg/ml was added to the H2O2 solution. fraction, LC50 = 260.89 μg/ml, hexane fraction, LC50
Decrease in absorbance of H2O2 at 285nm was = 333.91 μg/ml, ethyl acetate fraction, LC50= 45.85
determined spectrophotometrically 10 minutes later μg/ml, butanol fraction, LC50 = 2105.63 μg/ml and
against a blank solution containing the test extract in aqueous fraction 3152.61 μg/ml (Table 1). The result
PBS without H2O2. All tests were run in triplicates corroborated the presence in the plant of medicinally
and averaged [41,36]. The same experiment was active compounds. The use of Peperomia pellucida
carried out on Butylatedhydroxyanisole (BHA), is reported to be contraindicating in lactation as it is
ascorbic acid and α-tocopherol which are known also said to interfere with prostaglandin synthesis [1].
antioxidant standards. The toxicity result obtained in our assay also buttress
this observation. However the ingestion of the polar
Antioxidant activity by ferric thiocyanate method: fractions (aqueous and butanol) of Peperomia
pellucida may not be harmful to the human body due
The antioxidant activities of hexane, ethyl to its non-toxicity.
acetate and butanol fractions of the plant material

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