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Advances in Environmental Biology, 9(19) Special 2015, Pages: 50-56
AENSI Journals
Advances in Environmental Biology
ISSN-1995-0756 EISSN-1998-1066
Journal home page: http://www.aensiweb.com/AEB/
Corresponding Author: Ma. Reina Suzette B. Madamba, Department of Biological Sciences, Mindanao State University-
Iligan Institute of Technology. A. Bonifacio Ave. Tibanga, Iligan City. 9200. Philippines.
Tel: +6309177181101 E-mail: mrsmadamba_ps31@yahoo.com
Phytochemical and Antibacterial Properties of the Ethanolic Leaf Extract of
Merremia Peltata (L.) Merr. and Rubus SPP
Kristine Jay B. Perez, Mark Anthony I. Jose, Edgardo Aranico and Ma. Reina Suzette B. Madamba
Department of Biological Sciences, College of Science and Mathematics, Mindanao State University-Iligan Institute of Technology, A.
Bonifacio Ave., Iligan City, 9200 Philippines
A RT I C LE I NF O
A B ST R AC T
Article history:
Received 23 June 2015
Accepted 25 July 2015
Available online 30 August 2015
Keywords:
Antimicrobial activity, ethnomedicinal
plants, Kirby- Bauer Disk diffusion
method, Merremia peltata (L.) Merr.,
Rubus spp.
Rubus species have been cultivated and have been used traditionally for therapeutic
purposes as an astringent, ophthalmic and restorative. In the Philippines, the leaves of
this plant have been used as treatment for cough especially by the Higa-onon tribes in
Rogongon, Iligan City. While Merremia peltata (L.) Merr. is a species of flowering vine
in the morning glory family, Convolvulaceae and the leaves are used for stomach pains,
skin sores and inflammation. This study was to determine the phytochemical and
antibacterial properties of ethanolic and water leaf extracts of these two ethnomedicinal
plants, Merremia peltata (L.) Merr. and Rubus spp. since not much has been known so
far. The Kirby- Bauer disc diffusion method was used to test the antimicrobial activity
of the two extracts at 5, 10, 15, 20 and 25 ug/ml concentrations. Four bacterial strains
were used and were grown overnight at 37°C in the Mueller- Hinton Agar (MHA)
plates with the treated discs. Results showed that both ethanolic and water extracts of
Merremia peltata (L.) Merr. and Rubus spp. had inhibitory effect on Gram-positive
(Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Escherichia coli and
Pseudomonas aeruginosa) bacteria. The ethanolic and water leaf extracts of both plants
showed strong to moderate inhibitory activity against the test bacterial strains compared
to positive controls. However, the inhibitory effect exhibited by ethanolic extract was
significantly higher than that by water extract (p<0.05). Also, the inhibitory activity of
both plant extracts on all test isolates showed no significant difference among
concentrations. Phytochemical analysis revealed the presence of steroids and flavonoids
in both plant samples. Alkaloids were found only in Merremia peltata (L.) Merr. while
tannins were only found in Rubus spp. Thus, this may indicate that both plants have the
potential bioactive substances for medicinal purposes as claimed by the Higaonon tribe.
© 2015 AENSI Publisher All rights reserved.
To Cite This Article: Kristine Jay B. Perez, Mark Anthony I. Jose, Edgardo Aranico and Ma. Reina Suzette B. Madamba, Phytochemical
and Antibacterial Properties of the Ethanolic Leaf Extract of Merremia Peltata (L.) Merr. and Rubus SPP. Adv. Environ. Biol., 9(19), 50-56,
2015
INTRODUCTION
Today, plant materials remain an important resource for combating illnesses, including infectious diseases
and many of these plants have been investigated for novel drugs or new therapeutic agents, food additives,
agrochemicals, and industrial chemicals [2]. Despite the increasing use of synthetic drugs, it persisted as the
“treatment of choice” since the therapy with synthetic antibiotics is not always possible because of their high
cost as well as toxicity due to their extended use [14]. The medicinal value of plants lies in some chemical
substances that produce a definite physiological action on the human body. The most important of these
bioactive compounds of plants are alkaloids, flavonoids, tannins and phenolic compounds [7].
Phytochemical analysis of medicinal plants has shown that numerous secondary compounds in plants
traditionally used for medicinal purposes have chemical properties effective at treating illness. This was
observed in the work done by Wadood et.al [20], on the phytochemical analysis of medicinal plants occurring
on local area of Mardan, where all extracts used have displayed anti-inflammatory, antianalgesic, anti-cancer,
anti-viral, anti-malarial, anti-bacterial and anti-fungal. Studies like this are important and have commercial
interest in both research institutes and pharmaceutical companies for the manufacturing of new drugs for
treatment of various diseases.
51 Ma. Reina Suzette B. Madamba et al, 2015
Advances in Environmental Biology, 9(19) Special 2015, Pages: 50-56
Moreover, there is a continuous and urgent need to discover new antimicrobial compounds with diverse
chemical structures and novel mechanisms of action because there has been an alarming increased in the
incidence of new and reemerging infectious diseases [16, 18]. There is therefore a need to search for more
herbal medicines with the aim of validating the ethno-medicinal use and subsequently the isolation and
characterization of compounds which will be added to the potential list of drugs [15].
This study used two plants specifically Merremia peltata (L.) Merr. and Rubus spp. which has been used
ethno-medicinally among the Higa-onon tribe of Rogongon Iligan City as - remedy to various illnesses. The
genus Rubus is comprised of more than 250 species of shrubs, often with prickles on stems and leaves within
this genus from the rose (Rosaceae) family. They are found throughout the world, some are cultivated for their
ornamental value, as a useful food source, while others are regarded as weeds [1]. Rubus spp. is a spreading,
low, spiny shrub, rarely exceeding a meter in height. Leaves are pinnate with 5 to 7 leaflets. Leaflets are smooth
or hairy, with lobed margins, oblong-ovate or broadly lanceolate, and 2 to 7 centimeters in lenght. Flowers are
borne upon slender, prickly, hairy, 3 to 5 centimeter-long stalks and arise from the uppermost leaf axils. Calyx is
hairy. Petals are white, broadly oblong, and constricted toward the base. Berries are red, conically elongated,
and 1.5 to 2 centimeters across.
Merremia peltata (L.) Merr. is under the family Convolvulaceae known as bindweed or morning glory
family. This plant is a coarse and widely spreading woody vine. Stems are 5 or more centimeters thick, and
porous. Leaves are alternate, smooth, somewhat rounded, about 20 centimeters wide, those toward the ends of
the branchlets being much smaller, heart-shaped at the base, pointed at the tip. Peduncle grows solitary from
each of the upper leaf axils, erect and longer than the leaves. Flowers are large, golden-yellow, few to many, or
clustered. Sepals are smooth, thick, oblong, 2 centimeters long. Corolla has a wide limb.
Thus, this study was conducted to determine their phytochemical property and investigate the bioactivity of
ethanolic and water leaf extracts of Merremia peltata (L.) Merr. and Rubus spp. through Kirby-Bauer disc
diffusion method against Gram-positive and Gram-negative bacteria inorder to assess their ethnomedicinal
viability.
MATERIALS AND METHODS
Collection of the Plant Samples:
The Merremia peltata (L.) Merr. and Rubus spp. plant samples (Fig. 1) were collected from Rogongon,
Iligan City during early morning. Disease free-looking and fresh plants were selected and its leaves were
carefully washed with tap water thrice. A total of 700 g plant samples were zip locked inside polyethylene bags.
Pressed samples were identified and authenticated at the Herbarium of the Department of Biological Sciences
Mindanao State University, Iligan Institute of Technology where voucher specimen was prepared and deposited.
The collected samples were hanged for days until sufficiently dried samples were obtained. The dried samples
were placed in polyethylene bags ready for further processing in the laboratory.
Preparation of the Plant Extracts:
Five hundred (500) grams of the two air-dried plant samples were pounded till its powder state was
achieved. Powdered samples were mixed in 100 ml 95% ethanol and enclosed in a sterile, dark container for 72
hours.
Phytochemical Screening:
The screening was done to determine the presence of bioactive chemical components in the two plant
samples such as alkaloids, antraquinones, cyanogenic glycosides, unsaturated sterols, flavonoids and saphonins
according to the standard protocols described previously [2, 6, 15].
Preparation of Microorganism:
In this study, pure cultures of four different pathogens (Staphylococcus aureus, Bacillus subtilis,
Escherichia coli and Pseudomonas aeruginosa) were obtained from the Philippine National Collection of
Microorganisms, National Institute of Molecular Biology and Biotechnology, University of the Philippines -
Los Baños. Prior to the antimicrobial activity study, the test microorganisms were subcultured on Nutrient Agar
(NA) media, incubated at 37oC for 24 hrs then inoculums of the test microorganisms were grown into Nutrient
Broth (NB) and adjusted according to Mac Farlands Standard to achieve approximately 1x108 CFU/ml before
inoculating to the test media, Mueller-Hinton agar (MHA).
Preparation of Controls:
Streptomycin and Chloramphenicol were used as the positive control for the Gram-positive (S. aureus and
B. subtilis) and Gram-negative (E. coli and P. aeruginosa) respectively. Standard dosage used in the study for
52 Ma. Reina Suzette B. Madamba et al, 2015
Advances in Environmental Biology, 9(19) Special 2015, Pages: 50-56
Streptomycin was 10µg/ml and for Chloramphenicol was 30µg/ml based on the Kirby Bauer Chart for
antibacterial susceptibility testing [10]. Ninety-five percent (95%) ethanol was used as the negative control.
Preparation of the Two Leaf Extracts as treatments:
The treatments used were 5µg/ml, 10µg/ml, 15µg/ml, 20µg/ml and 25µg/ml for both Merremia peltata (L.)
Merr. and Rubus spp. extracts with three replications. The solvent used for the preparation of the extract was
95% ethanol.
Antimicrobial Activity:
The Kirby-Bauer Disk Diffusion Method [12] which is commonly used method for antimicrobial activity
test was employed. Using the sterile micropippetor tips, 100 µL or 0.1 mL of bacterial culture in broth were
transferred to the MHA plates and was spread using a sterile L- rod asceptically to minimize contamination and
erroneous result. Sterile Whatmann’s No. 4 filter paper with a 6mm diameter was used as paper discs. The
specific filter paper discs with 10 µL each of the test extracts of various concentrations and the controls were
aseptically placed into the centre of divisions on each MHA plate using a forcep to test the activity of each
extract as an antimicrobial agent. The plates were incubated at 37oC for 24 hours. After incubation, zones of
inhibition were measured using a standard ruler by measuring the diameter of the clear zone in the nearest
millimeter. This was then subtracted with the diameter of the disc and the result was the measurement
considered in this study.
Data Analysis:
In order to determine whether different concentrations of the extracts were significantly different or not
with the controls, the average measurement of zone of inhibition for each concentrations were analysed using
Analysis of Variance (ANOVA).with 95% level of significance. Post-hoc test was also applied to assess
differences within treatments.
Results:
Phytochemical Sreening of the Ethanolic Leaf Extracts of Merremia peltata (L.) Merr. and Rubus spp:
Results of the phytochemical screening (Table 1) showed that only Merremia peltata (L.) Merr. leaf extract
had alkaloid while Rubus spp. extract had tannins. Both plants had more steroids and flavonoids except for the
two phytochemicals, anthraquinones and cyanogenic glycosides which were absent.
Antimicrobial Activity of the Ethanolic and Water Leaf Extract of Merremia peltata (L.) Merr. and Rubus spp:
A. Against Gram-positive (G+) Bacteria:
The Merremia peltata (L.) Merr. and Rubus spp. ethanolic and water leaf extract concentrations: 5µg/ml, 10
µg/ml, 15 µg/ml, 20 µg/ml, and 25 µg/ml had antimicrobial effect on the Gram-positive bacteria S.aureus and
B.subtilis as compared to the negative controls: ethanol and water. A mean zone of inhibition of 5.7 mm was
observed to be highly significant (p<0.05) for the ethanolic leaf extract of Merremia peltata (L.) Merr against S.
aureus and B. subtilis at concentrations 20 ug/ml and 10 ug/ml respectively (Fig.1A). For Fig.1B, the mean
zones of inhibition of 4.8 mm against S. aureus and 5.2 mm against B. subtilis were observed to be highly
significant (p<0.05) for the ethanolic leaf extract of Rubus spp. The observed results showed higher inhibitory
activity than that of the standard antibiotic Streptomycin which has a mean zone of inhibition of 2 mm.
Table 1: Phytochemical Screening Results of Merremia peltata (L.) Merr. and Rubus spp ethanolic leaf extracts.
Phytochemicals
Plant Samples
Merremia peltata (L.) Merr.
Rubus spp.
Alkaloids
( + )
( - )
Anthraquinones
( - )
( - )
Cyanogenic glycosides
( - )
( - )
Flavonoids
( ++ )
( +++ )
Steroids
( +++ )
( +++ )
Tannins
( - )
( ++)
Legend: (+) presence, (++) abundant, (+++) very abundant and (-) absence
B. Against Gram-negative (G-) Bacteria:
The measured zones of inhibition around the treated discs placed on the surface of the MHA plates showed
that the ethanol and water leaf extracts of Merremia peltata (L.) Merr. and Rubus spp. also inhibited the growth
of all Gram-negative bacteria. However, their antibacterial effectiveness varied and ethanol extract exhibited
more pronounced inhibition than water extract in the present study. Figure 2 showed that the various Merremia
peltata (L.) Merr. and Rubus spp. ethanolic and water leaf extract concentrations: 5µg/ml, 10 µg/ml, 15 µg/ml ,
20 µg/ml, and 25 µg/ml had antibacterial effect also on the Gram-negative bacteria E. coli and P. aeruginosa as
compared to the negative controls: ethanol and water.
53 Ma. Reina Suzette B. Madamba et al, 2015
Advances in Environmental Biology, 9(19) Special 2015, Pages: 50-56
Merremia peltata (L.) Merr. showed to be more effective in inhibiting Gram-negative bacteria than Rubus
spp. and had the most significant concentrations at 5µg/ml and 15µg/ml for E. coli and P.aeruginosa
respectively with mean zones of inhibition of 2.66 mm and 4.66 mm which was observed to be highly
significant (p<0.05) for the ethanolic leaf extract (Fig 2A). For Fig. 2B, the mean zone of inhibitions of 4.8 mm
against E. coli and 4.7 mm against P. aeeruginosa were observed to be highly significant (p<0.05) for the
ethanolic leaf extract of Rubus spp. at the same concentration of 25µg/ml. The observed results showed higher
inhibitory activity than that of the standard antibiotic Chloramphenicol which has a mean zone of inhibition of 1
mm at 30µg/ml.
Also, based on the results shown in Fig. 2 and 3 in comparing the inhibitory activity between ethanol and
water as solvent used for extraction, the solvent ethanol for both M. peltata and Rubus spp. plants showed higher
inhibitory activity than water extracts on all Gram-negative and Gram-positive microorganisms used. The
inhibitory activity of the ethanolic leaf extracts of the two plants was even higher than that of the positive
controls (Streptomycin in G+ bacteria and Chloramphenicol in G- bacteria) used.
Fig. 1: Merremia peltata (L.) Merr. leaf (left) and Rubus spp. leaves (right).
Fig. 2: Zone of inhibition of the ethanolic and water leaf extracts of (A) Merremia peltata (L.) Merr. and (B)
Rubus spp. against Gram- positive bacteria S. aureus and B. subtilis. Positive control is 10ug/ml
Streptomycin while 95% ethanol and water are the negative controls. The labels (***),(**),(*) mean
highly significant, more significant and significant respectively at p<0.05. Data is mean of 3 replicates.
54 Ma. Reina Suzette B. Madamba et al, 2015
Advances in Environmental Biology, 9(19) Special 2015, Pages: 50-56
Discussion:
Phytochemical analysis of medicinal plants has shown that numerous secondary compounds in plants
traditionally used for medicinal purposes have chemical properties effective at treating illness. Alkaloids are
nitrogenous compound that function in the defense of plants against herbivores and pathogens, and are widely
exploited as pharmaceuticals, stimulants, narcotics and poisons due to their potent biological activities. In
nature, alkaloids exists in large portions in the seeds and roots of plants and often in combination with vegetable
acids. Alkaloids have pharmacological applications as anesthetics and CNS stimulants [13]. In this study,
Merremia peltata (L.) Merr. leaves had an abundant amount of alkaloids but none was found in Rubus spp. The
presence of alkaloids in Merremia peltata (L.) Merr means that the plant could be used as anesthetics, CNS
stimulants, as antioxidant or free radical scavenger [11, 13]. However, in Rubus spp. there are more tannins
present but none in Merremia peltata (L.) Merr. Accordingly, these tannins have significant antibacterial
property which was observed in the work of Clark [3] and Ekpo and Etim [8]. Flavonoids and steroids were
found in both plants extracts. The former substance, flavonoids are chemical compounds active against
microorganisms. They have been found in-vitro to be effective antimicrobial substance against a wide array of
microorganisms [4, 9, 19]. This suggests that both M. peltata and Rubus. spp. may have an active antimicrobial
property.
Fig. 3: Zone of inhibition of the ethanolic and water leaf extracts of (A) Merremia peltata (L.) Merr. and (B)
Rubus spp. against Gram- negative bacteria E. coli and P. aeroginosa. Positive control is 30ug/ml
Chloramphenicol while 95% ethanol and water are the negative controls. The labels (***),(**),(*) mean
highly significant, more significant and significant respectively at P<0.05. Data is mean of 3 replicates.
The high presence of the latter component in the two plants, the steroids, is of importance and interest in
pharmacy due to sex hormones, corticosteroids and contraceptives [5, 17].
Merremia peltata (L.) Merr. and Rubus spp. leaf extracts inhibited the growth of all Gram-positive and
Gram-negative bacteria, but their effectiveness varied and ethanol extract exhibited higher inhibition than water
extract in the present study. This could be because ethanol being an organic solvent could dissolve organic
compounds better, hence liberate the active component required for antimicrobial activity since ethanol was
55 Ma. Reina Suzette B. Madamba et al, 2015
Advances in Environmental Biology, 9(19) Special 2015, Pages: 50-56
found easier to penetrate the cellular membrane to extract the intracellular ingredients from the plant material [9,
21]. Also, the presence of higher amounts of polyphenols in ethanolic extracts as compared to aqueous extracts
means that the former are more efficient in cell walls and seeds degradation which have nonpolar character and
cause polyphenols to be released from cells.
The ethanolic leaf extracts of both plants have higher inhibitory activity compared with the positive
controls, Streptomycin and Chloramphenicol against Gram+ (B. subtilis and S. aureus) and Gram- (E. coli and P.
aeruginosa) bacteria respectively. This suggests that both ethnomedicinal plants have pharmaceutical potentials
against wide arrays of microbes.
Conclusion:
The phytochemical screening of M. peltata (L.) Merr. and Rubus spp. ethanolic leaf extracts showed the
presence of flavonoids, steroids, alkaloids and tannins except tannins and alkaloids for the former and latter
plants respectively. Both ethanolic leaf extracts of the two plants have higher inhibitory activity compared with
the positive controls: Streptomycin and Chloramphenicol and the negative control. The study showed that the
two leaf extracts for both plants had inhibitory activity on both Gram-positive (S. aureus and B. subtilis) and
Gram-negative (E.coli and P. aeruginosa) bacteria however, the effect were varied as seen in the different
concentrations employed. Moreover, the ethanolic and not the water extract did show a significant inhibitory
activity against the different bacterial strains used. This is maybe attributed to the property of the ethanol as
organic solvent itself which could extract better the active intracellular ingredients from the plant material.
Overall, the results demonstrate and indicate that the Merremia peltata (L.) Merr. and Rubus spp. ethanolic and
water leaf extracts can be a source of bioactive substances that have a broad spectrum antimicrobial activity.
This confirms the traditional use of the leaves of Merremia peltata (L.) Merr. and Rubus spp. by the Higao-onon
tribes in Rogongon, Iligan City, Philippines for the treatment of diseases.
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