Phytochemical Screening and Antibacterial Activities of Bidens pilosa L. and Tridax procumbens L ...

23 Pages·2017·0.65 MB·English

International Journal of Modern Biology and Medicine, 2017, 8(1): 24-46
International Journal of Modern Biology and Medicine ISSN: 2165-0136
Florida, USA
Journal homepage: www.ModernScientificPress.com/Journals/IJBioMed.aspx
Article
Phytochemical Screening and Antibacterial Activities of Bidens
pilosa L. and Tridax procumbens L. on Skin Pathogens
Oluwole O. Owoyemi* and Muftau K. Oladunmoye
Department of Microbiology, Federal University of Technology, P. M. B. 704, Akure. Ondo state,
Nigeria
*Author to whom correspondence should be addressed; E-Mail: [email protected]
Article history: Received 29 July 2017, Revised 10 November 2017, Accepted 30 November 2017,
Published 6 December 2017.

Abstract: The antibacterial activities of the plants used by Rattus norvegicus (brown rat) in
the construction of its nest were studied. The quadrat sampling results of various brown rats’
nests sampled revealed the use of Bidens pilosa L. and Tridax procumbens L. leaves by R.
norvegicus (Brown rat) to constructing its nest. Hence, these plants were harvested and
extracted with solvents of distinct polarities and the extracts were qualitatively and
quantitatively screened for phytochemicals which revealed the presence of tannins, alkaloids,
flavonoids, saponins and cardiac glycosides in both plants. The antibacterial activities of the
ethanolic, aqueous and chloroform extracts of these plants at different concentrations of 50
mg/mL, 100 mg/mL and 200 mg/mL were evaluated on clinical skin pathogens and their
corresponding type cultures using agar well diffusion method. The ethanoic extract of T.
procumbens at 200 mg/mL exhibited broad spectrum activities on all the test bacteria except
B. subtilis. The zones of inhibition of T. procumbens extracts on the test bacteria at
200mg/mL ranged from 5.00±0.00 mm to 15.20±0.20 mm, the lowest inhibitory effect was
observed on Escherichia coli, while the highest was observed on Staphylococcus aureus
ATCC 43300. It was observed that the Gram positive organisms were more susceptible to
the extracts compared to the Gram negative organisms. However, B. pilosa extracts had more
antibacterial activities on Gram negative organisms compared to Gram positive organisms.
The results of this study revealed the therapeutic potentials of these plants selected by the
brown rat’s in controlling pathogenic microorganisms from its environment especially skin-
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Int. J. Modern Biol. Med.2017, 8(1): 24-46 25

related infectious microorganisms such as S. aureus evaluated and can also serve as a basis
for controlling the adverse effect of these pathogens in humans too.
Keywords: Bidens pilosa, Tridax procumbens, Rattus norvegicus, Phytochemical,
Antibacterial activities, Clinical, Type, Pathogens.

1. Introduction
Human is surrounded by countless microorganisms. The disease producing microbes play a very
important role in human life. Pathogenic microorganisms are always trying to develop resistance to the
various antimicrobial agents used for their control. Therefore, the chemotherapy of infectious diseases
has proved to be a continuous struggle. Scientists are always in search of new antimicrobial agents to
control the ever increasing menace of the microbes (Bushra and Ganga 2003). Thus, it is paramount for
microbiologists to conduct series of research even from the animal environments in order to seek for
alternative therapy for the resistant strains of infectious microbes.
Rat’s nest is a structure usually made up of plants material in which a rat lives and breeds its
young ones. There are different types of rat nest; each nest is always specific to a particular rat. Rats use
different types of leaves to constructing their nest depending on the specie and habitat of the rat
(Reichman and Smith, 1990). Roof rats find harborage in plants such as Algerian illy, bougainvillea and
dead fronds of palm tree. Wood rats sleeping nests were constructed of lichens, mosses, dried grasses,
shredded bark and wood and varied considerable in mass [30-215 g] (Hemmes et al., 2002). Wood rats
use bay leaf to control nest-borne ectoparasites (Hemmes et al., 2002). Bay leaf has a high
monoterpenoid content, which is noted for its biocidal activity, which has been shown to be toxic when
fed to laboratory mice (Hemmes et al., 2002).
Rats are specific about the plants they use in constructing their nests, using their instinct; they
carefully select plants that can confer anti-parasite and antibacterial activities which are used in
protecting themselves against insect herbivores (Langenheim, 1994) and pathogenic microorganisms
(Deans and Ritchie, 1987; Knobloch et al., 1989).Rat nest plays vital roles in the life span of a rat by
providing good health benefits to it such as regulating the nest temperature, protection against predators,
harborage and protection from infectious microbes.
Rattus norvegicus which is also referred to as brown rat, emóigbó in yoruba land (Nigeria),
Norway rat and street rat is one of the best known and most common rats. One of the largest muroids, it
is a brown or grey rodent with a body up to 25 cm long, and a similar tail length; the male weighs on
average 350 g and the female 250 g. Thought to have originated in northern China, this rodent has now
spread to all continents except Antarctica, and is the dominant rat in Europe, Africa and much of North
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America—making it by at least this particular definition the most successful mammal on the planet after
humans (Fragaszy et. al., 2003). With rare exceptions, the brown rat lives wherever humans live,
particularly in urban areas.
Selective breeding of R. norvegicus has produced the laboratory rat, a model organism in
biological research, as well as pet rats (Baker et al., 1979). Like mice, these rats are frequently subjects
of medical, psychological and other biological experiments, and constitute an important model organism.
This is because they grow quickly to sexual maturity and are easy to keep and to breed in captivity.
When modern biologists refer to "rats", they almost always mean R. norvegicus.
A 2007 study found brown rat’s to possess metacognition, a mental ability previously only found
in humans and some primates (Science Daily, 2007), but further analysis suggested they may have been
following simple operant conditioning principles (Smith et al., 2008).
The quadrat sampling results of various brown rats’ nests sampled revealed the use of Bidens
pilosa and Tridax procumbens leaves to constructing their nests. Previous studies on these plants show
that they belong to a class of medicinal plants (Dimo et al., 2001; Ali et al., 2001).
Medicinal plants are gifts of nature which are used to cure limitless number of diseases among
human beings (Bushra and Ganga, 2003). The abundance of plants on the earth’s surfaces has led to an
increasing interest in the investigation of different extracts obtained from traditional medicinal plants as
potential sources of new antimicrobial agents (Bonjar and Farrokhi, 2004).
Tridax procumbens L. belongs to the family of Asteraceae and commonly known as
‘Gaddichamanthi’ in Telugu, in Ayurvedic as Jayanthi, in Sidda/Tamil asVettukkaaya-thalai, in Folk as
Akalakohadi and in English as Coat buttons/Mexican Daisy, because of the appearance of its flowers
and is an ethno botanically important medicinal plant. The plant has been considered as a gregarious
weed, distributed throughout the tropics and sub tropics. It has been extensively used in Indian traditional
medicine as anticoagulant, antifungal and insect repellent; in bronchial catarrh, diarrhea and dysentery
(Ali et al., 2001).
T. procumbens is extensively used in the Indian Ayurvedic system of medicine for the treatment
of diarrhea, as an insect repellent, hair tonic and wound healer, i.e. the leaf juice is used to check
hemorrhage from cuts and bruises (Bhat et al., 2007). It is a well-known remedy for liver disorders and
has been shown to possess antidiabetic activity (Bhagwat et al., 2008).
B. pilosa is an erect, perennial herb widely distributed across temperate and tropical regions. B.
pilosa is either glabrous or hairy, with green opposite leaves that are serrate, lobed, or dissected. It has
white or yellow flowers, and long narrow ribbed black achene’s (seeds). It grows to an average height
of 60 cm and a maximum of 150 cm in favorable environments (Alcaraz and Jimenez, 1988).
B. pilosa, either as a whole plant or different parts, has been reported to be useful in the treatment
of more than 40 disorders such as inflammation, immunological disorders, digestive disorders, infectious
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diseases, cancers, metabolic syndrome, wounds, and many others (Pereira et al., 1999; Dimo et al.,
2001). B. pilosa is usually ingested; however, it can also be utilized externally. For instance, fresh B.
pilosa is used to treat snake bites and wounds (Dharmananda, 2013), and in Trinidad and Tobago the
aqueous solution of the leaves of B. pilosa is used to bathe babies and children (Lans, 2007).
This research investigated the phytochemical components in the two plants (B. pilosa and T.
procumbens) selected by the brown rat in constructing its nest as well as the antibacterial activities of
these plants on selected human skin pathogens.
2. Materials and Methods
The identification of the plants used by R. norvegicus in constructing its nest was investigated.
The physical observations of various newly-made nests of the brown’s rat and quadrat sampling of the
plants in the specified area of the rat nest region were carried out. The species of plants adopted by the
brown rat in constructing its nest were identified and authenticated by Prof. Y. A. Awodun in the
Department of Crop, Soil and Pest Management, The Federal University of Technology, Akure (FUTA),
Nigeria which were: Bidens pilosa L. and Tridax procumbens L.
2.1. Collection of Rattus Norvegicus Nest-leaves Composition, Preparation of the Extracts and
Percentage Recovery of the Extracts
The plant materials used by R. norvegicus in constructing its nests (B. pilosa and T. procumbens)
were collected from the nest environment in the outskirt bush of Edo-lodge Street Oke-ijebu Akure,
Ondo state. Having harvested the leaves, they were washed with tap water and air-dried at room
temperature (25±2ºC) for two weeks. The dried leaves were pulverized by grinding machine (type N
model) into smooth powder and subsequently sieved using 1.18mm sieve.
The method of Green (2004) was utilized for the extraction of the plants using solvent to sample
ratio of 10:1 (v/w). Two hundred grams portions of the pulverized leaves were separately weighed and
soaked in 2500 mL ethanol, aqueous and chloroform at ambient temperature for 72 h under regular
shaking condition. The extracts were filtered using Whatman filter paper. The filtrates were recovered
by removal of the solvents using rotary evaporator under reduced temperature at 40ºC.
The recovery rate of extracts was calculated using the formula below;
WA
% Recovery of extract = 𝑥 100
IW
Where WA = Weight of extracts recovered after extraction, IW = Initial weight of extracts.
2.2. Qualitative Phytochemical Screening of B. pilosa and T. procumbens
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The phytochemical analysis for qualitative detection of total flavonoids, tannins, alkaloids,
saponins, phlobatannin, cardiac glycosides, steroids, terpenoids and anthraquinone were performed on
the extracts as described by AOAC (2007).
Determination of saponin
Exactly 0.5000 g of the plant extract (B. pilosa or T. procumbens) was mixed with distilled water
in a test tube and shook together, frothing which persist on warming was taken as preliminary evidence
for the presence of saponins, absence of frothing on warming indicates negative result (AOAC, 2007).
Determination of tannin
Exactly 0.5000 g of the extract was stirred with 100 mL of sterile distilled water, filtered and
ferric chloride reagent was added to the mixture, a blue-black green or blue green precipitate was taken
as evidence for the presence of tannin while absence of a blue-black coloration indicates negative result
(AOAC, 2007).
Evaluation of phlobatannin
Deposition of red precipitate when 0.5000 g of the extract was boiled with 1% aqueous HCl was
taken as evidence for the presence of phlobatannin while the absence of red precipitate indicated negative
result (AOAC, 2007).
Determination of flavonoid
A 0.5000 g of the extract was stirred with 20 mL of dilute ammonia solution, a yellow coloration
was observed, and the disappearance of the yellow color after the addition of 1 mL concentration of
H SO indicated the presence of flavonoid while the presence of yellow coloration after the addition of
2 4
1 mL concentration of H SO indicated negative result (AOAC, 2007).
2 4
Determination of terpenoid
A 0.5000 g of the extract was mixed with 20 mL of chloroform and filtered. 3 mL of concentrated
H SO was added to the filtrate to form a layer. A reddish brown colour at the interface was observed
2 4
which indicated the presence of terpenoids while the absence of a reddish brown coloration indicated
negative result (AOAC, 2007).
Determination of cardiac glycosides
Exactly 1.000 g of the extract was dissolved in pyridine and few drops of 1% sodium
nitroprusside with few drops of 20% NaOH were added. A deep red coloration which faded to a brownish
yellow indicated the presence of cardiac glycosides while the presence of a deep red coloration which
does not fade away to a brownish yellow indicated the absence of cardiac glycosides.
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2.3. Quantitative Phytochemical Analysis of B. pilosa and T. procumbens
The quantitative analysis of the two plant extracts were carried out according to the method
described by AOAC (2007).
2.4. Collection of Test Microorganisms
Both clinical and type microorganisms were used for the study. The clinical organisms which
include: Staphylococcus aureus, Streptococcus pyogenes, Bacillus subtilis, Pseudomonas aeruginosa,
Escherichia coli and Klebsiella pneumoniae were obtained from Don Bosco Catholic Hospital Akure,
Ondo state, Nigeria and their corresponding type organisms were also obtained in Pathcare, Lagos State.
These were: Staphylococcus aureus ATCC 43300, Streptococcus pyogenes ATCC 29212, Bacillus
subtilis ATCC 21332, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 35218 and
Klebsiella pneumonia ATCC 48891.
2.5. Standardization of Inoculums
The inocula were prepared from the stock cultures which were maintained on nutrient agar slant
at 4OC and sub cultured onto nutrient broth aseptically using sterile inoculating loop and incubated at
37OC for 18-24 hours. The setup were suspended in saline solution (0.85% NaCl) and adjusted with the
aid of a spectrophotometer (Unico 1100RS) to match a turbidity of 0.5 McFarlands standard at
wavelength of 540 nm according to the method described by CLSI (2010).
2.6. Antibacterial Assay of B. pilosa and T. procumbens Leaf Extracts on Bacterial Test Organisms
Antibacterial activities of extracts were determined by agar well diffusion method as described
by Esimore et al. (1998) with slight modification. After standardization using 0.5 Mcfarland standard of
the inoculum, sterile Petri dishes were inoculated aseptically with 0.1 mL of the 18 hours old broth
cultures of the bacterial test organisms each, while 15 ml of sterilized nutrient agar was poured
aseptically in the inoculated plates. The plates were swirled carefully for even distribution and allowed
to gel. With the aid of a sterile cork borer of 6 mm in diameter, wells were made on the solidified agar
plate aseptically. A concentration of 50, 100 and 200 mg/mL of the extracts were prepared using 30%
dimethylsulphoxide (DMSO) as the reconstituting solvent and sterilized using 0.2 µm sterile membrane
pore filter paper. Using micropipette, each extract of 0.1 ml was then pipetted into the wells of
appropriately labelled plates and holes. The plates were allowed to stand on the laboratory bench for 15
minutes to allow proper in flow of the solution into the medium before incubating the plates at 37ºC for
24 hours. The control was prepared by using 0.1 ml of reconstituting solvent (30% DMSO) and incubated
alongside with the extracts plates. After incubation, the zones of inhibition (diameter) formed in the
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medium were measured in millimeter to determine antibacterial effectiveness of the extracts on the test
organisms. The experiment was carried out in triplicates.
2.7. Determination of Minimum Inhibitory Concentrations (MIC) of B. pilosa and T. procumbens
Extracts on Test Organisms
The tube dilution susceptibility test was used to determine the MIC values of the plant extracts
on the test organisms using the method of CLSI (2006). A series of Mueller-Hinton broth tubes
containing varying two fold concentrations of the various plant extracts in the range of 300 mg/mL to
12.5 mg/mL were prepared and incubated with a previously standardized density of the test organisms
(0.5 mL). The lowest concentration of the plant extract resulting in no growth following visual inspection
after 18-24h of incubation for bacteria was recorded as the MIC.
3. Results and Discussion
3.1. Percentage Recovery of Plant Extracts
The percentage recovery of each extract after the extraction processes was evaluated and shown
in table 1.
Table 1: Result of the percentage recovery of plant extracts
Solvent Original weight/input Extracted (%)recovery=
(g) weight/output(g) 𝐖𝐀
𝒙 𝟏𝟎𝟎
𝐈𝐖
A. Ethanol 250 16.00 6.40%
1. Ethanol 250 18.70 7.48%
B. Aqueous 250 11.20 4.48%
2. Aqueous 250 10.20 4.08%
C. Chloroform 250 9.70 3.88%
3. Chloroform 250 14.50 5.80%
Key: 1, 2, 3 = Bidens pilosa extracts; A, B, C = Tridax procumbens extracts. WA = Weight of extracts recovered after
extraction, IW = Initial weight of extracts.

The ethanolic extract of B. pilosa had the highest percentage recovery of 7.48%, followed by
chloroform extract with 5.80% and the least was observed on aqueous extract with 4.08% extracting
value. Moreover, the ethanolic extract of T. procumbens had the highest percentage recovery of 6.40%,
followed by aqueous extract with 4.48% extraction value and the least was recovered from chloroform
extract with 3.88% (Table 1).
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3.2. Qualitative Phytochemical Screening of B. pilosa and T. procumbens
The ethanoic, aqueous and chloroform leaf extracts obtained from B. pilosa and T. procumbens
were qualitatively screened for phytochemicals. As in table 2, the result of the phytochemical screening
revealed the presence of tannins, alkaloids, flavonoids, saponins and cardiac glycosides in both plants
[B. pilosa and T. procumbens].
Table 2: Qualitative Phytochemical screening of B. pilosa and T. procumbens

Phytochemical Extracts BEE BAE BCE TEE TAE TCE

Saponin + + + + + +
Tannin + + + + + +
Phlobatannin - - - - - -
Flavonoid + + + + + +
Steroid + + - + + -
Terpenoid + - - - - -
Cardiac glycoside + + - + + -
Alkaloid + + + + + +
Anthraquinone - - - - - -
Key: + = present, – = absent, BEE= Ethanolic extract of Bidens pilosa, BAE = Aqueous extract of Bidens pilosa, BCE =
Chloroform extract of Bidens pilosa, TEE = Ethanolic extract of T. procumbens, TAE = Aqueous extract of T. procumbens,
TCE = Chloroform extract of T. procumbens.

3.3. Quantitative Phytochemical Analysis
3.3.1. Quantitative phytochemical analysis of B. pilosa
The ethanolic, aqueous and chloroform leaf extracts obtained from B. pilosa were screened
quantitatively for phytochemicals. The results presented in fig. 1.
Tannin content had the highest value in ethanolic extract containing 23.50±0.45 mg/g, followed
by cardiac glycoside content of 20.52±0.58 mg/g. The phytochemical contents present in aqueous extract
ranges from 7.13±0.01 to 16.18±0.02 mg/g with steroids being the lowest and cardiac glycosides being
the highest. The chloroform extract of B. pilosa extract components consist saponin, tannin, flavonoid
and alkaloid with saponin content being the highest (15.63±0.38 mg/g) and flavonoid content being the
lowest (8.41±0.30 mg/g). The tannin content recorded was 12.63±0.00mg/g and alkaloid content was
12.97±0.01mg/g (Fig. 1).
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Fig 1: Quantitative phytochemical components of B. pilosa
3.3.2. Quantitative phytochemical analysis of T. procumbens
The ethanoic, aqueous and chloroform leaf extracts obtained from T. procumbens were
quantitatively screened for phytochemicals. The quantitative phytochemical analysis of T. procumbens
ethanoic extract displayed high flavonoid content compared to all other phytochemicals present
containing 25.54±0.25 mg/g followed by alkaloid content of 6.13±0.01 mg/g. The lowest value was
observed in aqueous extract of T. procumbens for tannin content of 2.83±0.01 mg/g, followed by steroids
content of 3.09±0.13 mg/g (Fig. 2).

Fig 2: Quantitative phytochemical components of T. procumbens
3.4. Comparative Antibacterial Activities of Bidens pilosa Extracts at 50 mg/ml on Test Organisms
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The antibacterial activities of crude ethanoic, aqueous and chloroform leaf extracts of Bidens
pilosa at 50 mg/ml concentration on clinical and type test microorganisms after 24 hours of incubation
are presented in Table 3.

Table 3: Comparative antibacterial activities of Bidens pilosa at 50mg/ml on test microorganisms
(Zones of inhibition, mm)

Organisms Source Ethanol Aqueous Chloroform Control
*E. coli 4.30±0.10b 0.00±0.00a 0.00±0.00a 0.00±0.00a

E. coli ATCC35218 3.13±0.20c 3.03±0.05c 2.13±0.15b 0.00±0.00a

*K. pneumoniae 2.36±0.15b 0.00±0.00a 0.00±0.00a 0.00±0.00a
K. pneumoniae ATCC48891 3.13±0.15c 0.00±0.00a 2.26±0.25b 0.00±0.00a
*P. aeruginosa 4.40±0.30c 2.36±0.25b 0.00±0.10a 0.00±0.00a

P. aeruginosa ATCC27853 4.96±0.57d 3.33±0.15c 2.36±0.25b 0.00±0.00a

*B. subtilis 0.00±0.00a 0.00±0.00a 0.00±0.00a 0.00±0.00a
B. subtilis ATCC21332 1.96±0.57c 1.00±0.00b 2.36±0.25d 0.00±0.00a
*S. aureus 3.20±0.10b 0.00±0.00a 0.00±0.00a 0.00±0.00a

S. aureus ATCC43300 5.16±0.15d 2.36±0.25b 3.20±0.10c 0.00±0.00a

*S. pyogenes 2.06±0.11b 0.00±0.00a 2.26±0.15b 0.00±0.00a
S. pyogenes ATCC29212 3.20±0.10d 2.20±0.10c 1.10±0.17b 0.00±0.00a
Values represent means ± standard deviation of triplicate readings. Superscripts of the same letter in a row are not significantly
different at P≤0.05.
*Clinical isolate
The zones of inhibition (mm) of the extracts on the test organisms evaluated ranges from
1.00±0.00 mm to 5.16±0.15 mm. The highest zone of inhibition was observed on Staphylococcus aureus
ATCC 43300 (ethanoic extract) with inhibition zone of 5.16±0.15 mm while the lowest zone of
inhibition was observed on Bacillus subtilis ATCC 21332 (aqueous extract) with inhibition zone of
1.00±0.00 mm (Table 3). It is noteworthy that the clinical test organism of B. subtilis was resistance to
all the extracts at this concentration. However, the clinical and type culture of Streptococcus pyogenes
were susceptible to the inhibitory effects of all the B. pilosa extracts at this concentration except for the
aqueous extract in which the clinical organism was resistance.
3.5. Comparative Antibacterial Activities of Bidens pilosa Extracts at 100 mg/ml on Test Organisms
The antibacterial activities of crude ethanoic, aqueous and chloroform leaf extracts of Bidens
pilosa at 100 mg/ml concentration on clinical and type test microorganisms after 24 hours of incubation
is presented in Table 4.
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