Asian Journal of Biological and Life Sciences Original Research

Antimicrobial Activities and Toxicities of the

Leaf Extracts of Ficus nota (Blanco) Merr.
Felmer S. Latayada1, Mylene M. Uy2*
1Department of Natural Sciences, Caraga State University, Butuan City, Philippines
2Department of Chemistry, Mindanao State University-Iligan Institute of Technology, Iligan City, Philippines
E-mail : mylene603@yahoo.com Contact No. : +63-63-2215041 loc 4123
Submitted : 25.07.2016 Accepted : 20.09.2016 Published : 30.12.2016
Abstract
The antimicrobial activities and cytotoxic potentials of the ethanol, hexane, chloroform and aqueous extracts of the Philippine medicinal plant F. nota
was evaluated using disc diffusion assay and brine shrimp lethality assay, respectively. The aqueous extract (FnA) showed active to very active levels
of inhibition against the three bacterial test organisms B. subtilis, S. aureus, and E. coli, with zones of inhibitions ranging from 18.7±1.15 mm to
20.0±0.00 mm. The extract FnA further exhibited activity against the two fungi S. cerevisiae (11.7±1.15 mm, partially active) and C. albicans
(14.0±1.00 mm, active). This significant antimicrobial results of the aqueous extract was followed by the chloroform extract, FnC (17.0±5.2 mm,
active against B. subtilis and 22.3±4.62 mm, very active against S. aureus). Meanwhile, the toxicity test showed that the ethanol extract FnE scored
significant toxicities against the test organism Artemia salina with LC50 values of 79.43 g/ml and 206.5 g/ml, respectively. This study shows that the
bioactive components of F. nota with antibacterial, antifungal, and potential cytotoxic properties were successfully and effectively extracted and
concentrated in the various leaf extracts. These findings further support the ethno-medicinal uses of F. nota.
Key words : medicinal plant, cytotoxicity, antimicrobial, anticancer, synergistic effects

INTRODUCTION nota is among the plants considered in this study. F. nota (Blanco)
Merr., a member of family Moracea, is a small tree endemic to the
I
[1,2] [3,4]
nfectious diseases and cancer are the leading causes of Philippines and locally known as “tibig”. The young leaves are
morbidity and mortality around the world and continue to used as vegetables when cooked and the ripe fruit can be eaten
present major public health concerns. Today, many infections are raw. It is used to treat fever by extracting the water from a standing
caused by multi-resistant microorganisms that have resulted to a tree and drunken daily and applied to ease muscle pain. The
very demanding work to treat the diseases, and consequently decoction of roots and bark and the water from cut branches are
increase the healthcare cost[1]. Likewise, the limited efficiency used to treat urinary tract infection, hypertension and diabetes[9]. A
and the high cost of existing cancer therapies such as radiation, chemical investigation on the dichloromethane extract of the
chemotherapy, and surgery are indications of the high mortality unripe fruits of F. nota afforded the following chemical
rate among cancer patients[4]. Nature provides a vast source of constituents: 4-(2-hydroxyethyl)-2-methoxyphenol, a mixture of
natural products[1] that has been historically used for treatment of meso-2,3-butanediol, (2R,3R)-2,3-butanediol and (2S,3S)-2,3-
various diseases, and researchers around the world have butanediol and β-sitosterol[9]. Cytotoxicity study using in vivo
capitalized and made significant progress in the discovery of new brined shrimp lethality of the decoction and ethanol extracts of the
antimicrobial and anticancer compounds[4]. Evidence of the stem of F. nota were active against the brine shrimp Artemia
success in natural product drug discovery is clear as more than salina with LC50 values of 991.00 ppm and 852.22 ppm,
80% of drug substances are natural products or inspired by a
respectively[10]. The decoction and ethanol extracts of the leaves of
natural product compound and over 100 natural-product-derived
F. nota has been recently reported to possess considerable
compounds are currently undergoing clinical trials with at least
antioxidant properties[11]. However, there are no reports yet on the
100 similar projects in preclinical development in all major
antimicrobial activities and potential cytotoxicities of the leaf
therapeutic areas[2,5]. However, despite of the successes, the anti-
extracts of F. nota. This study was, therefore, carried out to
infective field is experiencing a shortage of lead compounds
investigate the antimicrobial and cytotoxic efficacy of the leaf
progressing into clinical trials[2] to control the spread of drug- extracts of F. nota not only to establish the scientific basis for its
resistant pathogens[6]. Meanwhile, in cancer chemotherapy, many traditional utilization as herbal medicine but also with the hope of
cancer patients develop resistance to treatment with standard anti- providing vital information on the possibility of isolating
cancer agents and this has become a serious problem[7]. bioactive chemical compounds that can be used as drug or drug
Therefore, there is a pressing need for screening, isolation, and leads. Additionally, these plants extracts could possibly be used to
identification of new antimicrobial and anticancer agents from produce alternative forms of natural products that could act in
new sources including plants. synergism with conventional drugs to combat the growing threat
The Philippines has remarkable biodiversity and is rich in of multi-drug resistant pathogens in anti-infective and anti-cancer
traditions of plant use. However, scientific studies and therapies.
pharmacological investigations of Philippine medicinal plants MATERIALS AND METHODS
just gained momentum recently[8]. The importance of the country's
diverse medicinal plants lies not only in their chemotherapeutic Preparation of plant extracts
value in traditional healthcare but also on the great prospect of Fresh and healthy leaf samples of the F. nota were collected
finding a new source of bioactive chemical compounds[8]. Ficus within the surrounding area of Caraga State University,

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Ampayon, Butuan City, Philippines. Identification and Toxicity assay: Brine shrimp lethality test
authentication of the collected plant material was done by Prof.
Meljan T. Demetillo of the Dept. of Biology, College of Arts and Brine shrimp lethality bioassay[13,14] was carried out to
Sciences, Caraga State University, Philippines. Voucher specimen investigate the potential cytotoxicity of the leaf extracts of F.
is deposited at the laboratory of one of the authors. The fresh plant nota. The eggs of brine shrimp (A. salina Leach) was introduced
leaves were washed thoroughly under running water, air-dried for on the other side of the small compartmentalized tank with a
about two weeks, homogenized and stored in airtight containers black-tinted glass divider filled with filtered and sterile sea water,
until use. Appropriate amount of homogenized powdered plant covered with clean aluminum foil and fully aerated at room
materials were soaked in an adequate amount of 95% ethanol for temperature. Drops of yeasts solution were added to both sides of
72 hours. The resulting mixture was filtered, concentrated in the compartments to supply food for the resulting nauplii. The
other side of the compartment was left open to allow contact with
vacuo using rotary evaporator at temperature not exceeding 400 C,
air and illuminated. After 48 hours, the newly-hatched nauplii
and weighed to provide the ethanol extract. A portion of the crude
were attracted and moved to the illuminated compartment and
ethanol extract was then sequentially partitioned in hexane:water
were collected with a Pasteur pipette. Briefly, stocks solutions (10
and chloroform:water solutions. The hexane-soluble,
mg/ml) of all the plant fractions were prepared by dissolving
chloroform-soluble and water-soluble portions were then
them in their respective solvents. Different levels of
individually concentrated under vacuo and then weighed to get
concentrations (1000, 500, and 100 g/ml) were prepared by
the extracts of hexane, chloroform and water, respectively. The
drawing different volumes from the stock solutions and added
plant extracts were coded as follows: FnE (F. nota ethanol
into the test tubes, dried using high purity nitrogen gas, dissolved
extract), FnC (F. nota chloroform extract), FnH (F. nota hexane
in dimethyl sulfoxide (DMSO) (0.6%) and then added with 4 ml
extract), and FnA (F. nota aqueous extract).
filtered and sterilized sea water. Ten brine shrimps nauplii were
Microorganisms and culture media added to each of the test tube. The volume was then adjusted to 5
ml with filtered and sterilized sea water. Each level of
The microorganisms were obtained from the Department of
concentration was tested in triplicates. The negative control
Science and Technology of the Philippine Government. The test
contained brine shrimp, filtered and sterilized sea water and
microorganisms were as follows: (a) bacteria: Staphylococcus
DMSO (0.6%) only. All the prepared test tubes were maintained
aureus No. 4, Bacillus subtilis No. 122, Pseudomonas aeruginosa
under illumination and number of dead and alive larvae was
No. 5, Escherichia coli No. 78, and (b) fungi: Aspergillus flavus
counted after 24 hours. The toxicity results of the plant extracts
No. 3006, Aspergillus niger No. 5540, Candida albicans No.
were evaluated by calculating the percent mortality of the brine
2049, and Saccharomyces cerevisiae No. 2006. Bacterial cultures
shrimps using the equation,
were maintained on nutrient agar (NA) and fungi were maintained
on Sabouraud dextrose agar (SDA). % Mortality= (Total no. of dead brine shrimps) / (Total no. of
brine shrimps) x 100
Antimicrobial activity: Disc agar diffusion method
and the LC50 values were then determined using the Reed-
In vitro antimicrobial assay of the ethanol, hexane,
chloroform, and aqueous extracts of F. nota was done using disc Muench method.
agar diffusion method[12] with minor modifications. Microbial RESULTS
suspensions of specific test microorganisms were swabbed
separately onto plates containing nutrient agar (NA) for bacteria Antimicrobial activity
and Sabouraud dextrose agar (SDA) for fungi. Five minutes after The antimicrobial activity of the leaf extracts of F. nota
swabbing, sterile paper disc with 6 mm in diameter, previously against different representative test organisms (bacteria and
soaked separately in 10, 000- ppm concentration extracts, fungi) was evaluated and expressed in terms of zones of
negative control (solvents: ethanol, hexane, chloroform, and inhibition in mm. The results of this assay are presented in Tables
water), and positive control (bacteria: Himox (5 mg/ml) for B. 1 and 2.
subtilis and S. aureus, Maxitrol (1mg/3.5mg/6000IU) for E.coli
and P. aeruginosa, fungi: Sporanox (10 mg/ml) for A. flavus and Toxicity assay
A. niger, Nystatin (100,000IU/ml) for C. albicans and S. In this assay, the cytotoxicity of the leaf extracts of F. nota
cerevisiae) were placed in each agar test plate equidistant to each were measured against the brine shrimp A. salina and expressed
other by carefully dropping them in with a sterile pointed dental as percent mortality and LC50 values. Table 3 summarizes the
probe, and then pressing them on the agar very lightly. The paper results.
disc containing the test fractions and extraction solvents were
allowed to dry for a few minutes before introducing into the agar DISCUSSION
plates. The seeded agar plates with impregnated paper discs were Antimicrobial activity
stored for about 6 h in the refrigerator to allow diffusion of the
impregnated material onto the agar. The plates were then The F. nota leaf extracts showed varying levels of
incubated, upside down, 24 h (for plates seeded with bacteria) and antibacterial activities. The ethanol extract of F. nota (FnE)
48 h (for plates seeded with fungi) at 370 C after which the zones of showed very active of inhibition only against S. aureus.
inhibition were then measured. The measurement was done by Surprisingly, the aqueous extract FnA had a remarkable range of
taking the total diameter of the zone (including the disc within) in activity against the four test bacterial organisms. FnA was able to
mm, at 2 perpendicular diameters then the average was calculated. inhibit the three bacterial organisms E. coli, B. subtilis, and S.
Three trials with two replicates were done on each of the F. nota aureus with zones of inhibitions ranging from 18.7±1.15 mm to
extracts. 20.0±0.00 mm that fall under active to very active levels of
inhibitory activity. Moreover, the plant's chloroform extract, FnC
was active against B. subtilis and very active against S. aureus.

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Table 1: Antibacterial activity of F. nota leaf extracts and positive controls.

* - mean of 3 replicates and expressed as mean ± SD

** - positive control for Gram (+) bacteria
*** - positive control for Gram (-) bacteria
**** - Negative control
(–) - No activity
Nd - not determined
***** - <10 mm: inactive; 10 mm 13 mm: partially active; 14 mm 19 mm: active; >19 mm: very active

Table 2: Antifungal activities of F. nota leaf extracts and positive controls.

* - mean of 3 replicates and expressed as mean ± SD

** - positive control for fungi
*** - positive control for fungi
**** - Negative control
(–) - No activity
Nd - not determined
***** - <10 mm: inactive; 10 mm 13 mm: partially active; 14 mm 19 mm: active; >19 mm: very active

However, the plant hexane extract FnH exhibited no inhibition or controls showed no effect against the test bacterial organisms
activity against all the test organisms. Meanwhile, P. aeruginosa which suggest the efficacy of the plant extracts. The ethanol
was not inhibited by any of the plant extracts. The negative extract of the plant, FnE, has no activity against all the fungi test

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Table 3: Effects of F. nota leaf extracts on A. salina after 24 hour-exposure.

organisms. However, the aqueous extract FnW showed displayed toxicity (LC50 <1000 g/ml)[13] against the brine shrimp
inhibitions against two fungi. FnA was partially active against S. test. The crude ethanol extract FnE and the chloroform extract
cerevisea and active against C. albicans. FnA was inactive against FnC both scored LC50 values of less than 250 g/ml which are
A. niger and A. flavus. The negative controls showed no effect considered significantly active[17,18] and warrants further
against the fungi test organisms which suggest the efficacy of the investigation. Furthermore, the potency exhibited by these two
aqueous extract of F. nota (FnA). extracts are even higher than those of the decoction and ethanol
If active constituents are present in high amounts, it is possible extracts of the plant stem[10].
that other constituents exert antagonistic effects on the bioactive CONCLUSION
compounds[15]. It was observed that, to some extent, the hexane
extracted components (FnH) may have contributed antagonistic This study has shown that the aqueous and chloroform
effects[16] to the ethanol extract FnE on its antimicrobial activity extracts of the leaves of F. nota possess antibacterial, antifungal,
which resulted to the selective activity of FnE among the test and cytotoxic properties. These findings suggest that F. nota is a
organisms. The combined activity of all the components in the potential source of bioactive natural compounds.
ethanol extract of the plant was less than those expected for the ACKNOWLEDGMENTS
extracts partitioned from it. On the other hand, synergistic
effects[16] of the individual chemical components might be the The authors acknowledge the Commission on Higher
driving force that made FnA and FnC more effective. These plant Education Faculty Development Program II (CHED-FDP II) and
extracts (FnA and FnC) are still in crude state and they may the Department of Science and Technology-Philippine Council
contain two or more bioactive chemical components. These for Health Research and Development (DOST-PCHRD) of the
results indicate that the antimicrobial components of both plant Philippine government for the support.
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