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ABSTRACT Staphylococcus aureus and Escherichia coli are considered to be two of the most prevalent gram-positive and gram-negative bacteria, and the most frequent sources of bacterial infections and hospital-acquired diseases worldwide (Bachir raho and Abouni, 2015). This investigation examines the antibacterial properties of Bambusa vulgaris (Bamboo) leaves and Eryngium foetidum (Culantro) leaves against the microbial growth of S. aureus and E. coli in vitro, and determines the phytochemical constituents present in the ethanol extracts that could be responsible for the said mechanism of action. Phytochemical Analysis and Antibacterial Test – Paper Disc Diffusion Method were the standardized laboratory methods used in the study. Based on the result of phytochemical screening, the identified phytochemicals present in Bambusa vulgaris ethanol extract were alkaloids, quaternary bases, amines, unsaturated steroids, and saponins. On the other hand, Eryngium foetidum ethanol extract contains alkaloids, quaternary bases, amines, 2 deoxysugars, unsaturated steroids, and tannins. The antibacterial activity of B. vulgaris and E. foetidum extracts were evaluated using Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative). Zone of inhibition was observed in vitro via paper disc diffusion method. B. vulgaris reached 26mm on the first trial, 27mm on the second and 26 mm on the third trial with an average of 26mm against S. aureus, whereas in inhibiting the growth of E. coli bacteria, it reached 20mm growth inhibition from trials 1 to 3 respectively. The extract from the E. foetidum (culantro) leaves grasped 22mm on the first trial and 23mm on the second and third trial with an average of 23mm against S. aureus, while in E. coli it exhibited 20 mm of zone inhibition from first to second trial and 21 mm on the third trial with an average of 20mm. The results of the tests on both samples implicate that these plants could potentially be used as antibacterial agents and on other medicinal applications. These findings could provide a prognostic approach in terms of addressing the manifestations caused by the foregoing detriments.
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Global Journal of Medicinal Plant Research
2018 January;6(1): pages 6-13
DOI: 10.22587/gjmpr.2018.6.1.2
Research Article AENSI Publications
Global Journal of Medicinal Plant Research
ISSN:2074-0883
Antibacterial Properties of Bambusa vulgaris (Bamboo) Leaves and Eryngium foetidum
(Culantro) Leaves against Staphylococcus aureus and Escherichia coli bacteria
Marloie T. Menchavez*, Jerico D. Catipay, Aileen S. Espra, Pedrito M. Castillo II
1Senior High School Department, University of Mindanao, Bolton St, Talomo, Davao City, 8021 Davao del Sur, Philippines
2Department of Science and Technology, Caraga State University, Ampayon Campus National Highway, NH1, Butuan City, 8600 Agusan Del Norte, Philippines
Correspondence Author: Marloie T. Menchavez*. Senior High School Department, University of Mindanao, Bolton St, Talomo, Davao City, 8021 Davao del
Sur, Philippines.
E-mail: marloiemenchavez@gmail.com
Received date: 22 January 2018, Accepted date: 22 June 2018, Online date: 8 August 2018
Copyright: © 2018 Menchavez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Key words: Phytochemical Analysis, Bambusa vulgaris, Eryngium foetidum, Staphylococcus aureus, Escherichia coli, Antibacterial Test, Paper
Disc Diffusion Method
INTRODUCTION
Throughout the years, the number of cases associated with bacterial infections and hospital-acquired diseases have been
catastrophic. In fact, infectious diseases are included in the leading causes of mortality in the world (World Health Organization,
2017) and still increasing significantly. While health care has been one of the top priorities for governments worldwide, infectious
diseases still remain a major public health concern (Smith et al., 2016).
Henceforth, Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are considered to be the most frequent
sources of fatal infections and hospital-acquired diseases (Bachir raho and Abouni, 2015). Staphylococcus aureus is a gram-
positive coccus that induce multitudinous suppurative (pus forming) infections and superficial skin lesions such as boils, styes
and furuncles; serious infections include pneumonia, meningitis, phlebitis, mastitis, and urinary tract infections; and entrenched
infections including osteomyelitis and endocarditis. The pathogen could instigate food poisoning by releasing enterotoxins into
food, and superantigen-release in the blood stream causing toxic shock syndrome. Also, Escherichia coli is a gram-negative
bacillus that is commonly found in the digestive tract of humans and warm-blooded animals. While most strains of E. coli are
harmless, some types can cause several infections and illnesses such as Enterotoxigenic E. coli (ETEC) that is the major cause of
traveler’s diarrhea and other bacterial diarrheal illness; and Shiga toxin-producing E. coli (STEC), known as E. coli O157:H7 that
induces toxins causing severe foodborne disease and intestinal infection (World Health Organization, 2018; Madappa, 2017).
ABSTRACT
Staphylococcus aureus and Escherichia coli are considered to be two of the most prevalent gram-positive and gram-negative bacteria, and
the most frequent sources of bacterial infections and hospital-acquired diseases worldwide (Bachir raho and Abouni, 2015). This
investigation examines the antibacterial properties of Bambusa vulgaris (Bamboo) leaves and Eryngium foetidum (Culantro) leaves
against the microbial growth of S. aureus and E. coli in vitro, and determines the phytochemical constituents present in the ethanol
extracts that could be responsible for the said mechanism of action. Phytochemical Analysis and Antibacterial Test Paper Disc
Diffusion Method were the standardized laboratory methods used in the study. Based on the result of phytochemical screening, the
identified phytochemicals present in Bambusa vulgaris ethanol extract were alkaloids, quaternary bases, amines, unsaturated steroids, and
saponins. On the other hand, Eryngium foetidum ethanol extract contains alkaloids, quaternary bases, amines, 2 deoxysugars, unsaturated
steroids, and tannins. The antibacterial activity of B. vulgaris and E. foetidum extracts were evaluated using Staphylococcus aureus
(gram-positive) and Escherichia coli (gram-negative). Zone of inhibition was observed in vitro via paper disc diffusion method. B.
vulgaris reached 26mm on the first trial, 27mm on the second and 26 mm on the third trial with an average of 26mm against S. aureus,
whereas in inhibiting the growth of E. coli bacteria, it reached 20mm growth inhibition from trials 1 to 3 respectively. The extract from
the E. foetidum (culantro) leaves grasped 22mm on the first trial and 23mm on the second and third trial with an average of 23mm against
S. aureus, while in E. coli it exhibited 20 mm of zone inhibition from first to second trial and 21 mm on the third trial with an average of
20mm. The results of the tests on both samples implicate that these plants could potentially be used as antibacterial agents and on other
medicinal applications. These findings could provide a prognostic approach in terms of addressing the manifestations caused by the
foregoing detriments.
7
Citation: Marloie T. Menchavez., Jerico D. Catipay., Aileen S. Espra., Pedrito M. Castillo., 2018. Antibacterial Properties of Bambusa vulgaris
(Bamboo) Leaves and Eryngium foetidum (Culantro) Leaves against Staphylococcus aureus and Escherichia coli bacteria. Journal of Medicinal
Plant Research., 6(1): 6-13. DOI: 10.22587/gjmpr.2018.6.1.2
Anent to this, infectious diseases were estimated to have caused 4.6 million deaths worldwide in 2015 alone (World Health
Organization, 2017). In the Philippines, infectious diseases are included in the leading causes of death in all regions (Philippine
Statistics Authority, 2016; Department of Health, 2013).
The development of antibiotics had made a significant contribution to the global health community for decades, reducing
the incidence and prevalence of infectious diseases worldwide. However, the emergence of several multidrug-resistant pathogens
due to mutation (Klevens et al., 2007) has threatened the efficacy of these treatments (Bandow et al., 2003) and became a global
public health threat (Hogberg et al., 2010). Furthermore, the prognosis of the current problem is predicted to worsen, given that
there is a decline in the development of new antibiotic drugs, and the cases of bacterial infections are increasing. Ergo, there is an
urgent need to formulate new strategies to combat the proliferating cases of infectious diseases; one of which is to cogitate the
screening of medicinal herbs and plants for their antimicrobial properties.
Phytochemicals, which occur naturally in plants such as alkaloids, flavonoids, saponins, and tannins have generated
particular interest with regard to human health effects, specifically antibacterial activity (Aniszewski, 2007; Cushnie and Lamb,
2005; Scalbert, 2001; Arabski et al., 2011). Published literatures indicated that the aforementioned phytochemicals are functional
constituents present in several species of bamboo (Kaur et al., 2015; Durgesh and Tumane, 2014) and culantro (Okon et al., 2013).
Bambusa vulgaris or common bamboo is a fast growing and giant tropical clumping bamboo that could grow up to 10 -
20 m in height and 4 -10 cm in diameter. It is native to China and Madagascar, but is widely cultivated in many parts of the world.
The chloroform extract of its leaves is used to treat Mycobacterium tuberculosis, and tabasheer obtained from the culm internodes
is used for several medicinal applications, such as treating kidney troubles, bark anstringent, emmenagogue, infantile epilepsy,
fever and hematuria (Schröder, 2011). The effective ingredients of bamboo leaf include flavones, phenolic acid, lactones, amino
acids and micronutrients (Sahbuti, 2004). Worldwide, bamboo has made a mark in the field of functional and medical health food
with its rich antioxidant resource and specific functional factors (Goyal et al., 2010).
On the same vein, Eryngium foetidum or Culantro is a biennial herb native to continental tropical America and the West
Indies. It is commonly found in moist areas where heavy soils preponderate. It is also widely used in dishes throughout the
Caribbean, Latin America, and the Far East as a food flavoring and seasoning herb for a wide variety of foods. Culantro is
purportedly abundant in iron, carotene, calcium, and riboflavin. The plant is also used in a broad spectrum of medicinal
applications such as, treating fevers, chills, vomiting, diarrhea, pneumonia, flu, diabetes, constipation, colds, stomach pains, and
malaria fever (Honeychurch, 1980; Mahabir 1991; Ramcharan, 1999). The decoction from its leaves was also found to exhibit
anti-inflammatory properties (Sáenz et al., 1997).
Though several published studies have examined the antibacterial properties and phytochemical constituents of Bambusa
vulgaris and Eryngium foetidum, the data reports are still limited, specifically in the Philippines. Also, given the fact that these
plants are abundant in numbers; have been shown to exhibit antibacterial properties; and are cultivated in many parts of the world,
these could be used as an alternative treatment for numerous diseases and infections caused by S. aureus and E. coli bacteria.
Accordingly, the primary goal of this study is to evaluate the antibacterial activity of Bambusa vulgaris and Eryngium foetidum in
inhibiting the growth of the aforementioned strains of bacteria in vitro using the synthesized ethanol extracts from both plants’
leaves, distinctly. Investigating this area could provide a novel perspective in the global health industry in terms of addressing the
manifestations caused by the foregoing detriments. This study would also contribute to the body of knowledge, primarily in
instituting scientific bases to consider the use medicinal plants and herbs in developing and formulating therapeutic drugs to treat
numerous diseases and derangements.
METHODS
Phytochemical Analysis
Preparation and Extraction of the Plant Sample for Phytochemical Analysis
One kilogram of each plant sample was collected. The leaves were air dried for about one week to let the dews evaporate,
then cut and shredded into smaller pieces. The method is based from A Guidebook to Plant Screening: Phytochemical and
Biological Screening (Guevara, 2005). The method was used to do the phytochemical screening of the plant extract. One kilogram
dried ground plant is weighed in an Erlenmeyer flask. The sample was then treated with 80% ethyl alcohol to completely
submerge the sample. The stopper of flask was then placed and the material was soaked for 24-48 hours. The extract was filtered
through Buchner funnel with gentle suction. The flask and plant material was rinsed with fresh portion of alcohol. The washings
and plant material was transferred to the funnel, combined with the washings of the first filtrate. It was then applied with gentle
suction to complete collection of the plant extract. The plant residue was discarded. The filtrate was concentrated under vacuum at
temperature below 50°C to about 20 mL. The extract was then measured. The extracts of the plants were then stored in cold place
at 0°C to -5°C temperature.
Phytochemical Screening for Alkaloids
a. Preparation of Reagents
1. Mayer’s Reagent – 1.4 grams of Mercuric iodide (HgI2) is dissolved in 60mL of water. The mixture is then poured into a
solution of 5.0 grams of Potassium iodide (KI) dissolved in 10mL of water. This is then added with 100 mL.
8
Citation: Marloie T. Menchavez., Jerico D. Catipay., Aileen S. Espra., Pedrito M. Castillo., 2018. Antibacterial Properties of Bambusa vulgaris
(Bamboo) Leaves and Eryngium foetidum (Culantro) Leaves against Staphylococcus aureus and Escherichia coli bacteria. Journal of Medicinal
Plant Research., 6(1): 6-13. DOI: 10.22587/gjmpr.2018.6.1.2
2. Dragendorff’s Reagent
a. Solution A 0.85 grams of Bismuth nitrate is dissolved in a mixture of 10mL Acetic Acid and 40 mL of water.
b. Solution B 8 grams of Potassium iodide (KI) is dissolved in 20mL of water.
c. Stock Solution- Solution A and B is mixed with equal parts. The resulting mixture was stored in a dark bottle
for a long time at room temperature.
d. To prepare the Reagent Mix 1mL stock solution with 2mL Acetic Acid and 10 mL water.
3. Preparation of 2 molar Hydrochloric acid (HCl) - 17mL of concentrated Hydrochloric acid is added to enough distilled
water to make 100mL of solution.
Alkaloid Analysis through Laboratory Test Tube Method
20 grams of plant materials from the stock plant extract was taken and placed in an evaporating dish. This is then allowed
to evaporate to syrupy consistency over a steam bath. 5mL of 2 molar Hydrochloric acid is added and heated with stirring rod in 5
minutes and then cool. About 0.5 grams of Sodium chloride (NaCl) is added then stirred and filtered. The residue was washed
with enough 2 molar of Hydrochloric acid to bring the filtrate to a volume of about 5mL. 1mL of the filtrate is taken and tested
with 2 to 3 drops of Dragendorff’s Reagent and another 1 mL of the filtrate is taken and tested with 2 to 3 drops of Mayer’s
Reagent. The test result will then be confirmed with the confirmatory test.
Confirmatory Test
The remaining 3mL of the filtrate was added with 28 % ammonia until the solution is alkaline to litmus. This test was
done carefully because it may cause burns, and its vapors are extremely irritating. The alkaline solution was extracted 3 times with
small portions of less than 10 mL chloroform. The lower chloroform extracts were also combined. The upper aqueous layer was
reserved for quaternary base amine oxide test. The chloroform extracts were evaporated to dryness under the hood and over a
steam bath. The residue was taken up with 5mL of 2 molar Hydrochloric acid and stirred over a steam bath for 2 minutes and
cooled. The filtrate was filtered and divided into 2 portions. The 1 portion of it was tested with Mayer’s reagent and the other with
Dragendorff’s reagent. If the result was recorded as (++). Positive result was indicated in the test, which means, secondary
alkaloid is present in the plant extract.
Test for Quaternary Bases and Amine Oxide
The alkaline aqueous layer was acidified with 2 molar of Hydrochloric acid. The filtrates were then filtered and divided
into 2 portions. The first portion was tested with Mayer’s reagent and the other with Dragendorff’s reagent. If the result was
indicated as (+), that means negative for quaternary bases or it may suggest that the extraction of the plant extract is incomplete.
Testing for Steroids
This test was done to determine the 2-deoxysugars and the unsaturated steroids through Keller-Killiani Test and Liebermann-
Burchard Test respectively.
1. Preparation of Keller-Killiani Reagents
a. Iron (III) Chloride Reagent 3mL of 1 % Iron (III) chloride (FeCl3) is dissolved in 50mL glacial acetic acid.
b. 1 % Iron (III) Chloride solution 1.0 grams of Iron (III) chloride (FeCl3) is dissolved in 100mL water.
2. Test Procedure The one portion of the defatted aqueous layer free from hexane is added with 3 mL of Ferric chloride
and then stirred. The 1 mL concentrated Sulfuric acid (H2SO4) is added continuously to the test tube in an inclined
position from a pipette, letting the acid trickle down along the insides of the test tube. The mixture is then allowed to
stand upright and was observed for any coloration at the interface of the acid and the aqueous layer.
If the reddish-brown color that turns purple was observed. This result will be indicated as (+), which means 2-deoxysugar is
present in the plant extract.
The Liebermann-Burchard Test for Unsaturated Steroids
A portion of the defatted aqueous layer free from hexane was added with 10 mL of dichloromethane, stirred with a glass
rod for a few minutes and allowed to stand. The lower dichloromethane extract were pipetted off. The dichloromethane extract
was dried by passing the extract through 100mg anhydrous sodium sulfate that is placed over dry filter paper in a funnel. The
filtrate was divided into two portions which is the other one is used as control. The other portion was treated with 3 drops of acetic
anhydride. This is done carefully because it is very corrosive. This is then treated with one drop of concentrated Sulfuric acid
(H2SO4). It is then observed for immediate color change. The portion was allowed to stand for an hour and observed for further
color changes. The change of color is then compared to the control. The change of color that is brownish-green was not observed.
If this result was indicated (-), that means, the unsaturated steroid is present in the plant extract.
Test for Leucoanthocyanins
The method used to test the flavonoids is the Bate-Smith and Metcalf Method. 10 grams of plant material from the stock
plant extract is taken and allowed to evaporate to incipient dryness over water bath. This is then cooled in room temperature.
Defatting is done by taking up the residue with 9 mL hexane and water, in 2:1 ratio by volume. The hexane extract is then
discarded. The defatted aqueous layer is then defatted with 10mL of 80% ethyl alcohol. The filtrate was filtered and divided into
9
Citation: Marloie T. Menchavez., Jerico D. Catipay., Aileen S. Espra., Pedrito M. Castillo., 2018. Antibacterial Properties of Bambusa vulgaris
(Bamboo) Leaves and Eryngium foetidum (Culantro) Leaves against Staphylococcus aureus and Escherichia coli bacteria. Journal of Medicinal
Plant Research., 6(1): 6-13. DOI: 10.22587/gjmpr.2018.6.1.2
2 test tubes. The other portion is taken as control. The other portion is then treated with 0.5mL concentrated Hydrochloric acid.
Color is then observed if there’s a change. The treated portion is then warmed for 15 minutes in a water bath. It is then observed if
color is changed within an hour; the color is then compared with the control. If the result of this test was marked (+), that means
there is a presence of Leucoanthocyanins.
Test for Saponins
The froth test is used to determine if Saponins are present in a plant extract. A volume of plant extract equivalent to 2
grams’ plant material is taken and transferred to a test tube. 1 mL “gogo” extract in a separate test tube is prepared as the standard.
10mL of distilled water to each of the test tube is added, stopper and shook both test tubes vigorously in 30 seconds. The test tube
is allowed to stand for 10 minutes and was observed for “honeycomb” froth. The result of the plant extract was compared with
that of the standard. If the result of this test was marked (+), that means saponins is present in the plant extract.
Test for Tannins
Ferric Chloride Test is used to determine if the extract have tannins. The reagents prepared are the following: 10 % of
Sodium chloride by dissolving 10 grams of Sodium chloride in enough distilled water to make 100 mL solution, Iron (III) chloride
reagent by dissolving 3mL of 1 % of FeCl3 solution in 100mL distilled water, and 1 % solution of FeCl3 by dissolving 1 gram of
FeCl3 in 100mL distilled water. An equivalent of 10 grams of plant material from the stock plant extract is taken and allowed to
evaporate to incipient dryness over steam bath. The residue is then extracted with 20mL of hot distilled water and added with 5
drops of 10% Sodium chloride solution. The filtrate is then filtered and divided into 3 test tubes. One portion is taken as the
control. An aqueous solution of tannic acid is used as reference standard. One portion of the filtrate was treated with 3 drops of
FeCl3 reagent and also done the same to the tannic acid solution. The result was then compared to the control and the reference
standard. If none of the color indications is observed to the extract that means no tannins present in the extract.
Antimicrobial Test
Preparation of the Plant Extract
The test material was prepared free from extracting solvent in which plant extract are completely syrupy in consistency. Water
soluble extract are dissolved in normal saline solution. Normal saline solution is prepared by dissolving 0.85 grams of A.R.
Sodium chloride in 1000 ml. distilled water.
Test Organisms
Staphylococcus aureus (gram-positive bacteria) and Escherichia coli (gram-negative bacteria) was used as assay organisms.
These bacteria represent major group of microbial flora.
Preparation of the test organism or inoculum
For gram-positive and gram-negative bacteria, 0.5 Mc Farland standards was used. A 0.5 McFarland was used to adjust the
turbidity of inoculum prior to the cotton swabbing of the agar plates for the antimicrobial assays. The standard was prepared and
subjected to quality prior to use. This standard contains approximately 1.5 x 108 CFU/ml of the test organism.
1. Preparation of 0.5 McFarland Standard 0.5 ml of 0.048 molar of BaCl22H2O to 99.5 ml of 0.36 normality of Sulfuric
acid (H2SO4) THAT IS 1 % volume per. 5 ml was distributed into screw-cap tubes of the same dimension as those to be
used in preparing the culture suspension. These tubes were tightly sealed and stored in the dark at room temperature. The
turbidity standard was shaking vigorously on a mechanical vortex prior to use.
Preparation of the Nutrient Broth for Gram-Positive Bacteria or Gram-Negative Bacteria
The 1000 ml nutrient solution was prepared. A loop of bacteria from the culture slant was inoculated in 50 ml nutrient
broth. The bacteria were incubated to the culture broth for 18 to 24 hours at 35°C. The level of turbidity was observed which
signifies that there is microbial growth.
Adjusting the turbidity
Five (5) ml of the culture broth was aseptically transferred to sterile screw-capped tubes. The bacterial suspension was
the agitated on a vortex mixer and immediately compared against the prepared 0.5 ml Mc Farland standard.
Preparation of the plates
Two plates were prepared for the Staphylococcus aureus and Escherichia coli. Approximately 15 ml melted nutrient agar
was poured into the dry and sterile petri dishes. The medium was allowed to solidify. A sterile cotton swab was moistened into the
test organism suspension. This was done through a wooden applicator handles. The sterile cotton swab was dipped into a
suspension of the test organism. The moistened swab was then pressed and rotated firmly against the inside side wall of the tube
just above the fluid level to remove the excess liquid.
Cotton Swabbing
The test organism was swabbed aseptically into a solidified nutrient agar by streaking the swab over the entire surface of the agar
plate 3 times. The plate was rotated 60° after each application to ensure an even distribution of the inoculum on the surface of the
medium. The swabbed plates were allowed to stand for about 5 minutes before the paper disc with the plant extract was applied.
Application of the plant extract through Paper Disc Diffusion Method
One paper disc was picked using forceps to immerse it into the plant extract. The disc was then placed gently on the agar
plate. The disc was then gently tapped, this is to ensure that the disc have a full contact with the agar medium. The plate was then
incubated in 48 hours.
Reading the Assay Plate
10
Citation: Marloie T. Menchavez., Jerico D. Catipay., Aileen S. Espra., Pedrito M. Castillo., 2018. Antibacterial Properties of Bambusa vulgaris
(Bamboo) Leaves and Eryngium foetidum (Culantro) Leaves against Staphylococcus aureus and Escherichia coli bacteria. Journal of Medicinal
Plant Research., 6(1): 6-13. DOI: 10.22587/gjmpr.2018.6.1.2
The clearing around the paper disc was then observed. This clearing around the paper disc is known as the zone of
inhibition. The plates were the inverted and the diameter of each inhibition zone was then measured in millimeters. The result was
expressed in (mm) and then recorded.
Bases of Analyzing the Antimicrobial Results
The analysis of the activity of the plant extract to the growth of bacteria was determined. Based on the standards, if the
clearing or “halo” around the paper disc measured lesser than 10mm (<10mm) is expressed as inactive, 10mm to 13 mm inhibition
zone is partially active, 14mm to 19 mm inhibition zone is active and greater than 19mm (>19mm) is considered very active
(Guevara, 2005). RESULTS AND DISCUSSION
Phytochemical Analysis
The phytochemical screening results of the ethanol extracts of B. vulgaris and E. foetidum are summarized in Table 1.
Phytochemical analysis for Bambusa vulgaris identified the presence of tertiary alkaloids, quaternary bases, amines, unsaturated
steroids, and saponins. On the contrary, Eryngium foetidum (culantro) leaves were observed to have the following phytochemicals,
namely primary alkaloids, quaternary bases, amines, 2 deoxysugars, unsaturated steroids, and tannins. Also, 2-deoxysugars was
only present in B. vulgaris extract, while saponins was only identified in E. foetidum extract. However, leucoanthocyanins was not
found in both ethanol extracts.
The findings from a previous study conducted by Owolabi and Lajibe (2015), showed that chloroform, n-hexane and
ethyl acetate extracts from B. vulgaris contain alkaloids, terpenoids, flavonoids, and tannins, however phenols was absent in both
chloroform and n-hexane extracts, but was present in ethyl acetate extract. Additionally, preliminary phytochemical screening on
the aqueous extract of B. vulgaris discovered the presence of alkaloids, flavonoids, saponins, terpenoids, steroids, coumarins,
phytosterols, and protein (Joselin et al., 2014). Besides, a review study instigated by Paul et al. (2011), stated that chemical
evaluation on E. foetidum leaves revealed the presence of flavonoids, saponins, tannins, and several triterpenoids. The absence of
other phytochemicals was possibly affected by the organic solvent used in the test, owing to the fact that different organic solvents
have distinctive capabilities in extracting biochemical constituents from a sample (Iwalokun et al., 2007). Furthermore, several
studies highlighted the antibacterial properties of alkaloids against gram-positive and gram-negative bacteria (Cushnie et al.,
2014), including S. aureus and P. aeruginosa (Mabhiza et al., 2016). Various reports have also shown that some alkaloids increase
the antibacterial activity of antibiotics (Zuo et al., 2011; Mitchell et al., 2012; Lavigne et al., 2010; Marquez et al., 2005; Maurya
et al., 2013) as cited by (Cushnie et al., 2014). Also, saponins extracted from Sorghum Bicolor L. Moench have been reported to
exhibit antibacterial activity against S. aureus bacteria in 50 and 25 mg/ml concentrations (Soetan et al., 2006). Overall, the result
denotes that B. vulgaris and E. foetidum contain phytochemicals that exhibit antibacterial properties against numerous pathogens.
Table 1: Phytochemical constituents present in the ethanol extracts of B. vulgaris and E. foetidum.
PHYTOCHEMICALS
Bambusa vulgaris
Eryngium foetidum
Alkaloids
(+) primary alkaloid
(++) secondary alkaloid
(+++) tertiary alkaloid
+++
+
Quaternary Bases and Amines
+
+
Steroids
-
+
+
+
Flavonoids
-
-
Saponins
+
-
Tannins
-
+
(-) = not present
(+) = present in small amount
(++) = present in moderate amount
(+++) = present in large amount
Antibacterial Test
The antibacterial activity of B. vulgaris and E. foetidum was examined using paper disc diffusion method. The results of
the antibacterial test on both extracts against S. aureus and E. coli bacteria are shown in Table 2. S. aureus and E. coli were found
to be susceptible to the ethanol extracts of both samples. Result confirmed that the growth inhibition of the extract from the B.
vulgaris (bamboo) leaves against S. aureus reached 26mm on the first trial, 27mm on the second and 26 mm on the third trial with
an average of 26mm, whereas in inhibiting the growth of E. coli bacteria, it reached 20mm growth inhibition from trials 1 to 3
respectively. The extracts from the E. foetidum (culantro) leaves reached 22mm on the first trial and 23mm on the second and
11
Citation: Marloie T. Menchavez., Jerico D. Catipay., Aileen S. Espra., Pedrito M. Castillo., 2018. Antibacterial Properties of Bambusa vulgaris
(Bamboo) Leaves and Eryngium foetidum (Culantro) Leaves against Staphylococcus aureus and Escherichia coli bacteria. Journal of Medicinal
Plant Research., 6(1): 6-13. DOI: 10.22587/gjmpr.2018.6.1.2
third trial with an average of 23mm against S. aureus while in E. coli it reached 20 mm from first to second trial and 21 mm on the
third trial with an average of 20mm.
Previous studies indicated that B. vulgaris has a wide scope of antibacterial activity. A research administered by Ambika
and Rajagopal (2017) showed that the acetone extract of B. vulgaris shoots, exhibit antibacterial activity against five strains of
bacteria, namely Klebsiella pneumonia, Staphylococcus epidermidis, Bacillus subtilis, Enterococcus faecalis, and Enterobacter
aerogenes. Two studies have also discovered that the dichloromethane extract obtained from the skin of bamboo shoots could
inhibit the bacterial growth of S. aureus (Tanaka et. al., 2011 and 2013). Likewise, n-hexane and ethyl acetate extracts of B.
vulgaris were found to demonstrate antibacterial activity against gram-positive bacteria Bacillus cereus and S. aureus (Owolabi
and Lajide, 2015). On the other hand, E. foetidum was also found to display strong inhibitive activity against gram-positive, gram-
negative bacteria and fungus. Lingaraju et al., (2016) showed that the ethyl acetate extract of E. foetidum inhibited the growth of
P. aeruginosa (28 mm) higher than that of the standard Gentamycin (18 mm); and exhibited a 25 mm, 17 mm, 20 mm, 18 mm
inhibition against S. aureus, E. coli, B. subtilis, and C. albicans. Moreover, revealed that methanol, petroleum ether, aqueous and
chloroform extracts of the same plant also exhibit antibacterial activity against some of the previously mentioned pathogens.
Table 2: Antibacterial activity of B. vulgaris and E. foetidum extracts using sterilized 6 mm paper disc diffusion method.
Zone of Inhibition (mm)
Trial
Bambusa vulgaris
Eryngium foetidum
S. aureus
E. coli
S. aureus
E. coli
1
26 mm
20 mm
22 mm
20 mm
2
27 mm
20 mm
23 mm
20 mm
3
26 mm
20 mm
23 mm
21 mm
Average
26 mm
20 mm
23 mm
20 mm
Table 3 indicates the standard list in analyzing the result of antibacterial test. Based on the standards, if the clearing or
“halo” around the paper disc measured lesser than 10mm (<10mm) it is expressed as inactive, 10mm to 13 mm inhibition zone is
partially active, 14mm to 19 mm inhibition zone is active and greater than 19mm (>19mm) is considered very active (Guevara et.
al., 2005). The average growth inhibition exhibited by B. vulgaris against S. aureus and E. coli bacteria is considered highly
effective, and the extracts of E. foetidum also display the same level of activity on both strains of bacteria. The results implicate
that B. vulgaris and E. foetidum could potentially be used as antibacterial agents.
Table 3: The standard list in determining the antibacterial activity of a sample.
Analysis of Result
Level of activity
Zone of Inhibition (mm)
Inactive
<10mm
Partially Active
10-13mm
Active
14-19mm
Very Active
>19mm
CONCLUSIONS
The study employed an in vitro method of examining the antibacterial activity of the ethanol extracts from Bambusa
vulgaris (Bamboo) leaves and Eryngium foetidum (Culantro) leaves. The researchers also identified the phytochemical
constituents present on both plants’ ethanol extracts, in which tertiary alkaloids, quaternary bases, amines, unsaturated steroids,
and saponins were identified to be present in the extracts B. vulgaris, while E. foetidum ethanol extracts were examined to have
following phytochemicals, namely primary alkaloids, quaternary bases, amines, 2 deoxysugars, unsaturated steroids, and tannins.
Furthermore, the two samples exhibited prodigious antibacterial activity against the growth of Staphylococcus aureus and
Escherichia coli bacteria. B. vulgaris exhibited an average growth inhibition of 26 mm against S. aureus and 20 mm on E. coli
which are both categorized as highly active. In addition, E. foetidum reached an average of 23 mm and 20 mm on both aforesaid
pathogens respectively. The results imply that these plants could potentially be used as antibacterial agents and on other medicinal
applications. RECOMMENDATION
12
Citation: Marloie T. Menchavez., Jerico D. Catipay., Aileen S. Espra., Pedrito M. Castillo., 2018. Antibacterial Properties of Bambusa vulgaris
(Bamboo) Leaves and Eryngium foetidum (Culantro) Leaves against Staphylococcus aureus and Escherichia coli bacteria. Journal of Medicinal
Plant Research., 6(1): 6-13. DOI: 10.22587/gjmpr.2018.6.1.2
The researchers of the study have identified the main flaw of this investigation, which is the lack of identification into
which of the following phytochemical constituents are accountable for the inhibitive activity of the two samples, though the result
of the phytochemical screening was shown. Thus, the researchers would highly recommend conducting further analysis on the
ethanol extracts of both plants and identify which of the isolates exhibit the identified antibacterial activity. In addition, the use of
other organic solvents should also be employed for the purpose of determining the extent of B. vulgaris’ and E. foetidum’
therapeutic properties.
ACKNOWLEDGEMENTS
The authors would like to extend their utmost gratitude to the people who have made significant contributions in this
work, most importantly, Jennifer J. Dejarme from the Department of Science and Technology, Caraga State University, Ampayon
Campus, for sharing her expertise in conducting the laboratory tests and to the administration of University of Mindanao for
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