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Toxicity and antioxidant properties of the extracts of Prunus grisea (C. Muell.) Kalkmleaves

Toxicity and antioxidant properties
of the extracts of Prunus grisea (C. Muell.) Kalkmleaves
Over decades, traditional medicine which includes the use
of herbal plants has been gaining widespread economic
and health system importance . According to the World Health
Organization, most of the population of many developing
countries regularly use remedies based on plants as their main
form of healthcare . The Philippines together with Hong Kong,
Malaysia, Mongolia, Singapore, Thailand, and Australia, are
classified as a countries “supportive” of traditional medicine
according to the World Health Organization. This implies that the
government recognizes the role played by traditional medicine,
supports its proper use, initiates efforts to bring proven traditional
medicine into the formal health service system and takes
measures to control its safe practice .
Prunus grisea (C. Muell.)Kalkm is identified as native plant
variety in the Philippines, Malaysia, Singapore, Taiwan and
China . This plant was reported to contain saponins, triterpenes
and steroids in a phytochemical survey of 212 plant species in
Malaysia . Detailed literature information on this plant is scant
but since the Subanen indigenous people of Southern Philippines
considered it as having therapeutic properties then, further
investigations can be done concentrating on its potentials as a
healthcare product. These include determination of its toxicity
and antioxidant capabilities in order to provide facts regarding its
application as herbal medicine.
The in vivo lethality in a simple zoological organism, such as
the brine shrimp lethality test can be used as a simple tool to
evaluate physiologically active plant extracts, where one of the
simplest biological responses to monitor is through lethality .
This general bioassay detects a broad range of biological activities
and a predictive method used for identifying cytotoxicity and
pesticidalactivity .
Reactive oxygen species (ROS) are commonly produced as
by-products of biological reaction or from exogenous factors,
Submitted : 12.04.2015 Accepted : 03.06.2015 Published : 30.08.2015
Decoction and various extracts (hexane, chloroform and aqueous) were prepared from the leaves of P. grisea. These extracts were subjected to
evaluation of toxicity using the brine shrimp lethality test and their antioxidant properties were determined different methods namely the 1,1-
Diphenyl-2-picrylhydrazyl (DPPH) radical- scavenging test, the phosphomolybdenum method for the total antioxidant capacity test and the Folin-
Ciocalteu method for the total phenolics test. The chloroform extract (PgC) exhibited the highest toxicity to the brine shrimp Artemia salina with LC
value of < 10 ppm. Results also indicated the decoction (PgD) of P. grisea leaves to have the most potent antioxidant properties, with percent
antiradical activity of 92.85% at 500 ppm, total antioxidant capacity of 110.42 Ascorbic Acid Equivalents (AAE) and 147.14 ButylatedHydroxytolene
Equivalents (BHTE) and total phenolics content of 319.00 Gallic Acid Equivalence (GAE). These data may help establish the medicinal potential of
this plant and may serve as a guide for further biological and chemical investigations on this plant.
Asian Journal of Biological and Life Sciences Original Research
Key words : medicinal plant, radical scavenging, total phenolics, phosphomolybdenum
Mylene M. Uy*, Princess T. Pundogar
Department of Chemistry, College of Science and Mathematics,
Mindanao State University-Iligan Institute of Technology, 9200, Iligan City, Philippines.
E-mail : Contact No. : +63-63-2215041 loc 123
which includes superoxide radicals, hydroxyl radicals, singlet
oxygen and hydrogen peroxide . Some of these ROS
positively function in vivo, such as in energy production,
phagocytosis, regulation of cell growth and intercellular
signaling or synthesis of biologically important compounds .
However, these species can also attack lipids in cell membranes
and DNA . These attacks lead to oxidations causing membrane
damage such as membrane lipid peroxidation, decrease in
membrane fluidity and DNA mutation leading to cancer .
Potential scavengers of ROS may provide possible preventive
intervention for free radical-mediated diseases . Recent
studies have shown that certain plant products including
polyphenolic substances (like the flavonoids and tannins) and
various plant or herb extracts exhibit antioxidant actions .
Antioxidants possess higher oxidative potential which can
protect or act as chain inhibitors of radical inducted
decomposition. Antioxidants break up the propagation chains by
acting as a hydrogen atom or an electron donor to the free radical
and as an acceptor of the excess energy released by the activated
molecule . At present, there is a growing interest on finding
naturally occurring antioxidants to be utilized in foods and
medicinal materials in replacement of synthetic antioxidants
which are being regulated because of their reported side effects
such as carcinogenicity . Antioxidants derived from natural
resources possess versatility in terms of multitude and magnitude
of activity .
There are many different methods for determining
antioxidant function which rely on different generators of free
radicals, acting by different mechanisms. Presently, a
combination of methods is being suggested in assessing
antioxidant activities in vitro in order to cover all the aspects of
antioxidant activities . The 1,1-Diphenyl-2-picrylhydrazyl
(DPPH) assay is known as one of the widely used test in
determining the scavenging activity of natural compounds due to
its low-cost and simplicity . In the DPPH assay, the antioxidants
are able to reduce the stable radical DPPH to non-radical form,
Asian J. Biol. Life Sci. | May-Aug 2015 | Vol-4 | Issue-2
DPPH-H. The color changes from purple to yellow after
reduction, which can be measured by its decrease of absorbance at
wavelength 517 nm. Radical scavenging activity increases with
increasing percentage of the free radical inhibition . The
phosphomolydenum method is known to be employed in
determining the total antioxidant capacity of plant extracts,
wherein the intensity of the green phosphomolybdenum complex
measured spectrophotometrically at 695 nm provides the
measure of total antioxidants found in the sample . Phenolic
compounds are widely and largely distributed in the plant
kingdom which are known to show multiple biological functions
including antioxidant activity . Polyphenols in plant extracts
react with specific redox reagents such as Folin-Ciocalteu reagent
to form a blue complex that can be quantified by visible-light
spectrophotometry . The content values are estimated by total
phenolic concentration equivalents of gallic acid, which is the
most important polyphenol in natural products .
Leaves of P.grisea were obtained from Mt. Kitanglad
Mountain Range, Bukidnon, Philippines. Authentication of the
plant's identity was done by the Department of Environment and
Natural Resources forester Mr.SeroñoSehagun of the Mt.
KitangladMountain Range Protected Area.In order to prepare the
crude extracts, about 5.0 kg of the fresh samples of the leaves was
properly washed in tap water and then rinsed in distilled water.
The rinsed sample was then initially air dried for two weeks. The
dried sample of the plant was pulverized using a sterile electric
blender, weighed and percolated with enough 95% ethanol for
three days. The solution prepared was filtered, concentrated in
vacuo at temperatures not exceeding 40 C to give the crude
ethanol extract. In hexane:water and chloroform:water solutions,
40 g of the crude ethanol extract was then sequentially
partitioned. The hexane-, chloroform- and water-soluble portions
was concentrated in vacuo and weighed to produce the hexane
(PgH), chloroform (PgC) and aqueous (PgA) extracts,
respectively. For the preparation of the plant decoction (PgD),
about 2.0 kg of fresh and clean leaves were incised into pieces and
boiled in sufficient amount of distilled water (1:2 ratio) for five
minutes. The mixture was then filtered, cooled and stored in glass
containers and freeze-dried.
The toxicity test of the various crude extracts was tested
against the brine shrimp Artemia salina leach following the
protocol previously described by Meyer et al and McLaughlin et
al with slight modifications. Four concentrations (1000-, 500-,
100-, and 10 ppm) were tested for each plant extract with three
replicates. Podophyllotoxin and the solvent served as the positive
and negative control respectively. The number of dead and alive
nauplii were monitored, counted, and recorded after 6 and 24
hours. Examination of the results and the determination of the
LC values were determined by employing the Reed-Muench
method .
The 1,1-Diphe nyl-2-picrylh ydrazyl (DPPH) radical
scavenging activity of the P. grisea extracts were evaluated by
com pari s on with t h e know n a ntio xida n t buty late d
hydroxytoluene (BHT) following the protocol as outlined by Lee
and Shibamoto . Each of the varying concentration of the
Plant Collection and Extraction
Toxicity assay using brine shrimp lethality test
DPPH radical scavenging test
extracts (500, 100, 50 and 25 µg/mL) was mixed with 3.0 mL of
methanolic solution of DPPH (0.1 mM). Then, the mixture was
vigorously shaken and allowed to stand at room temperature for
one hour. Using the spectrophotometer, the absorbance was
measured at 517 nm against methanol as blank. The percent of
DPPH decoloration of the samples was computed based on the
The evaluation of the total antioxidant capacity of the
different P. grisea extracts were carried out using the
phosphomolybdenum method described by Prieto et al .
Dispensed into 20 mL test tubes, 0.3 mL of the 200 µg/mL P.
grisea extract solutions were separately added with 3.0 mL
reagent solutions of 6M H SO , 28mM sodium phosphate and
2 4
4mM ammonium molybdate. Then, the test tubes were incubated
at 95 C for 90 minutes and then allowed to cool down at room
temperature for the absorbance measurement at 695 nm using a
spectrophotometer. The antioxidant activity was represented by
ascorbic acid equivalents (water-soluble components) and BHT
equivalence (fat-soluble components) that was derived from a
linear equation established using ascorbic acid and BHT as
reference standard. The results were recorded as means of three
trials performed.
The total phenolics of P. grisea extracts were determined
employing the method described by Makkar et al wherein 0.1
mL (0.5 mg/mL) of the extract was mixed with 2.8 mL of 10%
Na CO and 0.1 mL of 2N Folin-Ciocalteu reagent. The solution
2 3
was allowed to stand for 40 minutes and the absorbance was
measured at 725 nm. The total phenolic content was represented
as milligrams of gallic acid equivalence per gram of sample. The
calculations were based on the standard calibration curve derived
from the various concentrations of gallic acid (25-200 ppm) and
then the results were recorded as GAE (mg/g).
Table 1 presents the mortality rates of the brine shrimp A.
salina after 24 h exposure to different concentrations of the P.
grisea leaf extracts as well as the concentration of the extracts that
kills 50% of the brine shrimp (LC ).
The ability of the P. grisea leaf extracts to scavenge the free
radical DPPH are shown in Table 2.
Illustrated in Figure 1 are the total antioxidant capacities of
the P. grisea leaf extracts expressed as ascorbic acid equivalents
(AAE) and butylatedhydroxytoluene equivalents (BHTE).
Figure 2 shows the total phenolics content of the various
extracts of P. grisea leaves expressed as gallic acid equivalence
Total antioxidant capacity test by the phosphomolybdenum
Total phenolics content test
Toxicity assay using brine shrimp lethality test
DPPH radical scavenging test
Total antioxidant capacity by the phosphomolybdenum
Total phenolicscontent
Antiradical activity =
control - sample
A control
Asian J. Biol. Life Sci. | May-Aug 2015 | Vol-4 | Issue-2
Table 1: Toxicity of the various leafe xtracts of P. grisea to the brine shrimp A. salina.
* - mean of triplicate analysis
PgE Prunus grisea ethanol extract, PgH - hexane extract, PgC - chloroform extract,
PgA - aqueous extract, PgD decoction.
Table 2: DPPH radical-scavenging activities of P. grisea leaf extracts at various concentrations.
* - mean of triplicate analysis
** - butylated hydroxytoluene standard
PgE Prunus grisea ethanol extract, PgH - hexane extract, PgC - chloroform extract, PgA - aqueous extract,
PgD decoction.
Figure 1: Total antioxidant capacities of P. grisea leaf extracts at 200-ppm concentration
expressed as ascorbic acid equivalents (AAE) and buty. lated hydroxytoluene equivalents (BHTE)
Results show that the toxicity effects of the P. grisea leaf
extracts on the test animal A. salina was concentration
dependent. At 1000 ppm, PgH, PgC and PgA elicited a 100
percent mortality after a 24-hour exposure and by implication
1000 µg/ml of these P. grisea extracts is highly toxic to the brine
shrimp larvae. At 10 ppm, PgC still exhibited a relatively high
activity with 51.35% of the brine shrimps killed while the rest
were non-toxic anymore. Crude extracts with LC values of less
than 250 μg/ml are considered significantly active and have the
potential for further investigation for the possible presence of
bioactive components . In reference to this, PgC and PgA
extracts of P. grisea having the LC values of <10 ppm and
Asian J. Biol. Life Sci. | May-Aug 2015 | Vol-4 | Issue-2
Figure 2: Total phenolics content of P. grisea leaf extracts at 500-ppm
expressed as gallic acid equivalence (GAE)
199.53 ppm respectively, can be regarded to possibly contain
cytotoxic properties.
As shown by the results, the radical-scavenging activity of all
the P. grisea extracts increased as their concentrations were also
increased. The decoction (PgD) exhibited greater activity than the
control (BHT) at 50- and 100-ppm concentrations. However, it
showed almost similar radical-scavenging ability as the control at
500 ppm. The results also indicate that the decoction of the leaves
of P. grisea has high antioxidant activity compared with the
hexane-soluble, chloroform-soluble, ethanol-soluble and
aqueous extracts.
Meanwhile, all the extracts consistently showed the same
order of antioxidant capacity in terms of both AAE and BHTE
values i.e. PgD>PgH>PhE>PgC>PgA. The results also indicate
that the top four extracts (PgD, PgH, PgE, and PgC) contain
relatively more lipid-soluble antioxidants than water-soluble
The extracts displayed varying amounts of total phenolics
content with the decoction (PgD) having the highest value of
319.00 GAE and the aqueous extract (PgA) having the lowest at
65.25 GAE. The high GAE value of PgD suggests the presence of
substantial amount of phenolic compounds in this particular P.
grisea extract which supports its the strong DPPH radical
scavenging activity and its high total antioxidant capacity. Recent
investigations have shown that many flavonoids and related
polyphenols contribute significantly to the antioxidant activity of
many fruits, vegetables and medicinal plants .
The study has shown that the different extracts of P. grisea
leaves exhibited toxicity and antioxidant properties in varying
degrees and amounts. In reference to the results of the brine
shrimp lethality test, the chloroform (PgC) and aqueous (PgA)
extracts of P. grisea leaves having LC values of <10 ppm and
199.53 ppm respectively, are considered significantly active and
should be investigated further for the possible presence of
cytotoxic components. Over-all evaluation of the results of the
antioxidant assays strongly suggests that the decoction (PgD) has
the most potent antioxidant properties among the extracts of P.
grisea leaves. This indicates that bioactive molecules might be
present in PgD which can be used as a prototype for development
of new drugs and/or as a source of antioxidant pharmaceutical raw
The authors are grateful to the Science Education Institute and
Philippine Council for Health Research and Development of the
Department of Science and Technology of the Philippine
government for the financial and technical support provided for
the completion of the study.
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