BIBLIOGRAPHY MALEL, JOHN K....
BIBLIOGRAPHY
MALEL, JOHN K. APRIL 2013. Antibacterial activity of Bidens pilosa, Tithonia
diversofolia, Coix lacryma and Crassocephalum crepidioides. Benguet State University,
La Trinidad, Benguet.
Adviser: MYRNA B. WALSIYEN, MSc.
ABSTRACT
The study was conducted at the College of Veterinary Medicine, Benguet State
University, La Trinidad, Benguet to determine the antibacterial effects of Bidens pilosa,
Tithonia diversofolia, Coix lacryma and Crassocephalum crepidioides. Specifically it
aimed to show the positive and negative zone of inhibition of the four medicinal plants.
A total of 4 treatments were observed. Each treatment against the bacteria
Escherichia coli and Staphylococcus aureus.
The result of the study showed that there was no significant zone of inhibition
recorded on the gram positive bacteria Staphylococcus aureus. However, Tithonia
diversofolia recorded a positive zone of inhibition of 10mm. This indicated that the
medicinal plant Tithonia diversofolia water extract had antibacterial activity.
Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
INTRODUCTION
Many infectious microorganisms are resistant to synthetic drugs; hence an
alternative therapy is much needed. Medicinal plants have been used as an exemplary
source for centuries as an alternative remedy for treating diseases because they contain
numerous active constituents of immense therapeutic value.
Bidens pilosa locally known as burburtak is a wild crafted plant and is treated as
weeds in vegetable gardens in Benguet. Due to their abundance and reported biological
uses the researcher intends to test the antibacterial activity of water extracts of the plant
mentioned above against bacterial organism commonly found animals that can cause
diseases in man.
Crassocephalum crepidioides, also called “Ebolo” (in Yoruba land in Nigeria) is
an erect annual slightly succulent herb growing up to 180 cm tall. Its use is widespread in
many tropical and subtropical regions, but is especially prominent in tropical Africa where
the fleshy mucilaginous leaves and stems are eaten as vegetable and many parts of the plant
use for medical purposes. Medicinal folklore use of C. crepidioides include treatment of
indigestion (in Southern Nigeria), treatment of stomach upset (in DR Congo), treatment for
fresh wound (in Uganda), the decoction of the leaf is used in Nigeria for the treatment of
headache, in Tanzania a mixture of the leaf sap of C. crepidioides and Cymbopogon
giganteus is used orally and externally for the treatment of epilepsy. Also in Tanzania, the
dried leaf powder is applied as a snuff to stop nose bleeding and smoked to treat sleeping
sickness. Tannin found in the roots of the plant is used to treat swollen lips. The plant is
known to contain a large number of phytochemical compounds which include: tannin,
Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
dihydroisocoumarins, pyrrolizidine alkaloids such as jacobine and jacoline and
monoterpenes such as myrcene, limonene and α-copaene.
Tithonia diversifolia, also called wild sunflower is a small to medium-sized annual shrub
with rather stout, almost glabrous branches. Leaves are alternate, petioled,
membranaceous, ovate to orbicular, entire or 3- to 5-lobed, with toothed margins. It was
introduce to the Philippines as an ornamental, and has escaped cultivation to become a
weed in waste places.
In Central America, leaf extracts are used externally for the treatment of wounds
and hematomas. A methanol extract of the dried leaves of Tithonia diversifolia was
investigated for anti-inflammatory and analgesic activities. The extract produced dose-
related inhibition of carrageenan-induced paw oedema and cotton pellet-induced
granuloma in rats. At the same doses, analgesic effect was also observed with hot plate
latency assays maintained at (55 °C) as well as on the early and late phases of formalin-
induced paw licking in rats. The results of the present study further confirm the use of
Tithonia diversifolia traditionally for the treatment of painful inflammatory conditions
(Wuraola et al., 2004).
Job's Tears also known as Coix lachrymal has anti-inflammatory effects. Results
showed the methanol seed extract of seeds of CL showed anti-inflammatory properties
which may involved the inhibition of NO and O2 production by activated macrophages.
Study on the anti-inflammatory activities of six benzoxazinoids from roots of Coix
lachryma-jobi var. Ma-yuen showed the free hydroxyl group in the benzoxazinone skeleton
involved in the expression of inhibitory activity
Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
The main objective of the study is focused on the antibacterial activity of Bidens
ilosa, Crassocephalum crepiodioes jobs tears and Tithonia diversifolia extracts against
bacterial organism.
Specifically, it aimed to:
1. To determine the antibacterial activity of Bidens pilosa,Tithonia diversifolia,
Coix lachryma and Crassocephalum crepidioides, water extracts against Staphylococcus
aureus, and E.coli
2. To compare the antibacterial activity of water extracts on the inhibition of
bacterial organisms; and
3. To determine the best among the various treatments, Bidens Pilosa,
Crassocephalum crepidioides, Coix lachryma and Tithonia diversifolia, water extracts and
the positive control (Anpicillin) in the inhibition of bacterial organism.
Farmers will mostly benefit from the use of the above plants as an antibacterial
control for animal organism. It can sustain the productivity of the livestock industry in the
Cordillera through the promotion of good health and cheap alternative medicines for
animal diseases. Because of the abundance of this plant in the locality, they can be easily
utilized by farmers and other locals.
The study was conducted at the BSU College of Veterinary Medicine laboratory,
Balili, La Trinidad Benguet in February, 2013.
Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
REVIEW OF LITERATURE
Bidens pilosa
Bidens pilosa is called by various names, such as Spanish needle, Black jack or
Beggar ticks in English and “burburtak” in Ilokano, Kalinga and Bontok Mt. Province ;
“furfurtak” in Botbot and Kalinga; “Silew” in Bauko Mt. Province- Kankanai;
“Monmonot” in Sagada Mt. Province-kankanai; Onward or engguad in Ifugao; Nuad or
Ang-anguad in North Benguet or Puriket in Tuba- ibaloi.
It is an erect, branched, less hairy herb, about 0.2 to 1.5 m high. The leaves are two
pinnafid and 15 cm more or less, with the upper one being usually much smaller. The leaves
are ovate-lanceolate about 2-5 cm long and toothed. The flowers discs are brown, yellowish
or nearly whit flowering head about 8 mm long. The fruits achenes are black, long and
slender, linear, 1.0-1.5 cm long and characterized by four projections at the apex (Mvere,
2004).
The entire plant is common in waste places, from Batanes and Babuyan Islands and
Northern Luzon to Mindanao. The entire plant utilized usually as a decoction or poultice
in folkloric medicine against inflammation and as an external wash.
Bidens pilosa Folkloric Uses
In the Cordillera, the hot water decoction is used for the treatment of diarrhea,
prophylactic for gastrointestinal disorder, while it is usually taken with ginger decoction in
the treatment of gastric pains. The hot water decoction is also used as a foot bath for the
treatment of wound, inflammation. The sap from fresh leaves is mixed with honey for the
treatment of painful urination and acute nephritis.
Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
In many regions of Africa and tropical America, the roots, leaves and seed of Bidens
pilosa have been reported to possess antibacterial, antidysenteric, anti-inflammatory,
antimicrobial, antimalarial, diuretic, hepayo-protective and hypotensive activities (Mvere,
2004).
In Africa, the sap from crushed leaves is used to treat ear infection, speed up clotting
of blood in fresh wounds (Uganda); applied to burns (Tanzania). While the leaf decoction
is used for treating kidney problems (Uganda); cure stomach and mouth ulcers (Zimbabwe)
and treating arthritis (South Africa) and for treating jaundice (Cote d ‘Ivoire) and
suspension of powdered leaves as an enema for abdominal by Zulu in South Africa and
leaf extracts are used to treat swollen spleen of children by the Giriama tribe of Kenya. The
Giriama tribe also use a mixture of ground dried Bidens pilosa leaves, soap and hot pepper
for the control of leaf miners in plants (Mvere, 2004).
Bidens pilosa,Antimicrobial Properties
A number of studies have demonstrated the antibacterial activity of Bidens pilosa
against Staphylococcus aureus (Mvere, 2004). Klebsiella pneumonia, Bacillus, neisseria
gonorrhea, pseudomonas, and salmonella, Mycobacterium tuberculosis and M. smegmatis
(Mvere, 2004). The plant extracts is also effective against Candida and herpes virus
(Chiang et al., 2003).
The plant sap is bacteriostatic to Staphylococcus aureus but not to Escherichia coli
and the water extracts exhibit activity against intestinal pathogenic bacteria herpes virus
(Chiang et al., 2003). Ethanol and methanol extracts of roots of Bidens pilosa exhibit
Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
bacteriostatic effect against Staphylococcus aureus, Staphylococcus epidermidus
Escherichia coli and Kebsella pneumonia (Ashafa and Afolayayn, 2009).
However, water extracts of Bidens pilosa have no inhibitory property against
Staphylococcus aureus beta hemolytic streptoccus, Bacillus cereus and E. coli and
Pseudomonas and Candida (Rojas et al., 2006).
Bidens pilosa Phytochemical Analysis
Studies have shown that the plant contains iodine; the leaves, tannin and saponin;
the flowers, sulfur polyacetylene and flavonoid, and acetylene have been identified from
the plant extracts (Mvere, 2004). The entire plant contains Anthraquininone.
Phytochemical analysis of the plant showed that it is highly positive for gums,
mucilages and glycosides; carbohydrates and reducing sugars; tannin and tannin
derivatives and proteins and proteins derivatives. It is also positive for secondary
metabolites as steroids (unsaturated sterols) and deoxysugars; unsaturated sterols and
triterpenes and polyphenolic compounds
NSRU (2009) as cited by Battad (2009) reported that the phytochemical analysis of
the leaves of Coix lachrymal-jobi showed that it is highly positive for gums, mucilages and
glycosides; moderately positive carbohydrates and reducing sugars; highly positive for
tannin and tannin derivatives and proteins and protein derivatives. It is also positive for
secondary metabolites such as steroids (unsaturated sterols) and deoxysugars; unsaturated
sterols and titerpenes and polyphenolic compounds.
Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
Tithonia diversifolia Analgesic/Anti-Inflammatory
Study of methanol extract of dried leaves of Tithonia diversifolia produced dose-
related inhibition of carrageenan-induced paw edema and cotton pellet-induced granuloma
in rats. The analgesic effect was observed with hot plate latency assays. Results confirm
the traditional use of Tithonia diversifolia for the treatment of painful inflammatory
conditions (Wuraola et al., 2003).
Staphylococcus aureus
Staphylococcus aureus is a Gram – Positive spherical bacteria that occur in clusters
resembling grapes. It colonizes mainly the nasal passages, but may be found regularly in
the skin, oral cavity and gastrointestinal tract of man and animals. It cause superficial skin
lesions such as boils, stys and furuncles. It also causes food poisoning by releasing
anterotoxins into food and toxic shock syndrome by release of super antigens into the blood
stream (Todar, 2008). This bacterium has the ability to develop resistance against
antibiotics. MRSA are stains of the Staphylococcus that are resistant to the action of
methicillin and related beta-lactam antibiotics like penicillin, oxacillin and amoxicillin.
Battad (2009) reported that leaves of Bidens pilosa and Coix lachrymal in ethanol
extract have very active antibacterial effect on Staphylococcus aureus and E. coli.
Cogasi (2006) mentioned that the “ange” or the camote leaves and vines is the main
feed given to pigs by the people in the municipality of Besao. When the time comes that
it becomes scarce, most especially during summer time or during the dry season, the swine
raisers would use the other indigenous feeds, taro of which are the Bidens pilosa and
Crasophalum crepedioides or the Japanese weed.
Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
METHODOLOGY
Materials
The materials used in the study include leaves of Bidens pilosa, or “burburtak,
Crassocephalum crepidioides or Japanese weeds, Tithonia diversifolia or wild sunflower,
and Coix lachryma or Jobs tears. The test organisms were gram positive bacteria:
Staphylococcus aureus and Escherichia coli. The media that was used in the study was
Muller Hinton agar (MHA and sterile distilled water). Ampicillin disc was the positive
control against gram positive. The equipments that were used in the study were Ohaus
digital weighing scale, hot oven sterilizer biological incubator, auto clave, rotary vaporizer,
hot plate and laminar flow.
The glass wares used in the study include sterile beakers, graduated cylinder,
Erlenmeyer flask, stirring rod and vials. Other materials used in the study were sterile
cotton balls and swabs, sterile rubber gloves, denatured alcohol, alcohol lamp, match,
inoculating loop, wire basket, aluminum foil, scissors, ball pen, ruler, marker, notebook,
camera, and computer.
Methods
Experimental treatments. There were four treatments involved in the study. Each
treatment was replicated three times. The different treatments were as follows:
T0 – Antibiotics disc (Ampicillin)
T1 - Bidens pilosa leaves
T2 – Crassocephalum crepidioides leaves
Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
T3 – Tithonia diversifolia leaves
T4 – Coix lachryma leaves
Preparation of plant extract. Fifty grams (50g) leaves of each of the plants namely
Bidens pilosa, Crassocephalum crepidioides, Tithonia diversifolia and Coix lachryma were
weighed separately. These plant leaves were gathered at Balili, La Trinidad, Benguet.
These were washed and air dried. The leaves were cut using a sterile knife on a chopping
board and placed in the beaker. One hundred ml distilled water was added then heated and
was allowed to simmer for 10 minutes (Figure 1) The decoction was then decanted and the
liquid was transferred to another sterile container and was set aside (Figure 2). A sterile
filter paper disc (Whatman 10 ash less” filter paper) was soaked into the decoction
overnight. The Kirby-Bauer technique for determining antibacterial activity of the plants
used in the study was used.
Figure 1. The researcher heating the plants Figure 2. The decanted decoctions
Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
Inoculation of Mueller Hinton agar plate. Using a sterile cotton swab, the tip was
slightly moisten with 0.5 McFarland standard turbidity of test organism was used in
nutrient broth culture to Muller-Hinton agar plates. Then using sterile forceps, the disc
soaked in the plants’ extracts were placed on the agar plates (Figure 3). The same was
done with the antibiotic disc. The inoculated agar plates were wrapped with paper and
incubated at 35oC for 24 hours.
Preparation of filter paper. The filter paper discs were made by cutting Whatman
Ash-less filter paper No. 42 with a puncher to make 6mm diameter discs. The filter paper
discs was wrapped in a clean aluminum foil and sterilized in a hot oven sterilizer for an
hour in 100oC (Figure 4) The sterile filter paper discs were then soaked overnight into the
plant decoctions.
Figure 3. Inoculation of Mueller Hinton agar plate
Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
Figure 4. The researcher sterilizing the filter paper discs
Preparation of assay plates. Mueller-Hinton agar was prepared following the
manufacturer’s recommendations based on weight that was 3.07 grams per 100 ml of
distilled water. This was sterilized for 15 minutes the agar was then dissolved in 100 ml
distilled water.
Definition of Terms
1. Incubation. This is the maintenance of temperature, humidity and other
conditions while microbiological culture is being grown to multiply.
2. Zone of inhibition. This is the area where the growth of the bacteria is restricted.
The interpretation of the zone of inhibition measurement, as cited by Battad (2009), is as
follows:
Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
Zone of Inhibition (mm)
Interpretation
Less than 10
Inactive
10-13
Partially active
14-19
Active
Above 19
Very active
Data Gathered
1. Zone of inhibition of the water extracts or decoctions derived from the plants
and the positive and negative control on the gram positive bacteria. This was measured at
the widest zone of inhibition using a ruler or caliper.
Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
RESULTS AND DISCUSSION
Zone of Inhibition
Staphylococcus aureus. Results of the study revealed no recordings on the zone of
inhibition were obtained in all the plant extracts against Staphylococcus aureus (Table 1).
This means that the water extracts of the four plants namely Bidens pilosa or “burburtak”,
Crassocephalum crepidioides or Japanese weed, Tithonia diversifolia or sunflower and
Coix lachrymal or Jobs tears have no inhibitory properties against Staphylococcus aureus.
Ampicillin, the control, had also a zero zone of inhibition.
The above result is similar to the findings of Rojas et al (2006) who also found out
that water extract of Bidens pilosa has inhibitory properties against Staphyloccus aureus.
However, the result is in contrast to the findings of both Chang (2003) and Mvere (2004)
who both reported that the plant extract or plant sap of Bidens pilosa is bacteriostatic or
has antibacterial activity against Staphylococcus aureus.
Battad in 2009 also found out that both the Bidens pilosa and even Coix lachryma
are very active (23.33 and 25.33mm zones of inhibition, respectively) in inhibiting
Staphylococcus aureus but this was using ethanol extract. Ahmad and Ismail (2003)
reported that the water extract of Coix lachryma is used to treat fever and cough but
unfortunately it did not come out to have antibacterial effect with both Staphylococcus
aureus and Escherichia coli.
According to Cogasi (2006), Bidens pilosa and Crassocephalum crepidioides are
two of the common garden weeds fed by the swine raisers in Besao, Mountain Province to
their animals. These are normally cooked before feeding to the pigs. The pigs are
Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
Table 1. The zone of inhibition of the water extracts of the plants used in the different
treatments
ZONE OF INHIBITION
TREATMENT
Staphylococcus aureus Escherichia coli
Ampicilin (Control)
0
0
Bidens pilosa
0
0
Tithonia diversofolia
0
10 mm
Crassocephalum crepidioides
0
0
Coix lachrymal
0
0
Water extract of Tithonia diversofolia (sunflower) has pronounced inhibitory against E
coli.
usually fed with camote leaves and vines (the most commonly used feedstuffs for
swine) and this constitute the major part of the ration. However, in times when there is
scarcity of this camote leaves and vines, Bidens pilosa and Crassocephalum crepidioides
can serve as substitutes. While there is no scientific basis, it could be possible that the
giving of boiled Bidens pilosa and Crassocephalum crepidioides in greater amounts could
have helped in preventing illness to the pigs caused by Staphylococcus aureus.
Escherichia coli. Results of the study also showed that the water extracts obtained
from the leaves of Bidens pilosa, Crassocephalum crepidioides, and Coix lachryma had no
antibacterial activity against Escherichia coli as revealed by the zero zone of inhibition.
However, for the Tithonia diversofolia or wild sunflower, results of the study gave a
reading of 10 mm on zone of inhibition against Escherichia coli. Though the 10mm zone
of inhibition obtained was considered the minimum to have an antibacterial effect as cited
by Battad (2009), perhaps when the concentration or level of the wild sunflower leaves is
Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
increased in producing the water extract, its antibacterial activity is increased also. Among
the leaves used in the study, the wild sunflower is very bitter in taste. This means that it
has a component not present in the other leaves used which has an antibacterial activity.
The above result is similar to the findings of Chiang (2003) and Rojas et al (2006)
who both found out that the plant sap or water extract of the leaves of Bidens pilosa is not
bacteriostatic or has no inhibitory properties to Escherichia coli. On the other hand, Battad
(2009), in her study, found out that both the Bidens pilosa and Coix lachryma are very
active on inhibiting Escherichia coli but she made used of the ethanol extracts of both.
Figure 5. Zone of inhibition
Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
The Crasocephalum crepidioides, Coix lacrymai and the Bidens pilosa water
extracts does not have any antibacterial activity against Escherichia. coli and S. Aureus, it
enhances the ability of the immune system.
Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
SUMMARY, CONCLUSION AND RECOMMENDATION
Summary
This study was conducted to evaluate the antibacterial properties of plants namely
Bidens pilosa or “burburtak”, Crassocephalum crepidioides or Japanese weed, Titonia
diversofolia or wild sunflower and Coix lachrymal or Jobs tears agains against
Staphylococcus aureus and Escherichia. coli.
The plants used in the study were collected at Balili, La Trinidad, Benguet. The
plants composed the treatments as follows: T0 – Ampicillin; T1 – Bidens pilosa; T2 –
Crassocephalum crepidioides; T3 – Tithonia diversifolia and T4 – Coix lachrymal.
Fifty grams of each of the plants in the treatments were chopped and used in the
study. This was diluted with 50 ml distilled water to produce the water extract to be tested.
Results of the study revealed that there were no significant antibacterial properties
of all the plants used in the study except the Tithonia diversifolia or wild sunflower. There
was a positive zone of inhibition of the wild sunflower against Esherichia coli.
Conclusion
Based on the results of the study, it can be concluded that the Bidens pilosa,
Crassocephalum crepidioides, and Coix lachrymal water extracts do not have any
antibacterial effects against Staphylococcus aureus and Escherichia coli. The Tithonia
diversifolia water extract has no antibacterial effect against Staphylococcus aureus also but
has an antibacterial effect against Escherichia coli.
Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
Recommendation
It is recommended that further studies should be conducted to include higher levels
of the plant leaves in the water extract; their antibacterial effects on other bacterial
pathogens aside from Staphylococcus aureus and Escherichia coli and to include other
ways of extraction.
Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
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Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.
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Antibacterial activity of Bidens pilosa, Tithonia diversofolia, Coix lacryma and
Crassocephalum crepidioides | MALEL, JOHN K. APRIL 2013.