Antimicrobial Efficacy of Carabao Grass (Paspalum conjugatum) leaves on Staphylococcus

aureus
Dani Aiko Garduque, Kyrie Radiance Mateo, Shalom Moyinoluwa Oyinloye, and Jo Anne Kristine Lalas-
Lucero, Student of Adventist University of the Philippines
INTRODUCTION
In the Philippines, this variation of grass is Paspalum conjugatum, known by its common name as
“carabao grass”. Just like the other types of grass in other countries, carabao grass is mainly used as
forage for livestock. The name “carabao” grass is after the word carabao, which is a type of buffalo from
the Philippines. Goats, horses, sheep, cows and even carabaos graze on this type of grass.
Carabao grass is important for plantations as well, specifically in the rice and coconut plantations.
This is due to the allelochemicals in the carabao grass, which produces biochemicals that positively
affect the growth and reproduction of other bio-organisms (Kobayashi, 2004). For other uses, carabao
grass is used by landscaping companies as landscape material for malls, parks, and other recreational
areas. So far, there is little evidence that carabao grass is used as an antimicrobial agent in the
Philippines.
METHODS
Test for Glycosides (Fehling’s Test) - The plant extract was dissolved in hot water. After the
dissolution of the extract, the solution was filtered. The resulting filtrate was utilized for examination. Two
ml of the filtrate was placed in two test tubes for a total of 4 ml of the filtrate. One ml dilute HCL was
added in one of the test tubes, while the other test tube did not have any other additives. The test tube
that only had the filtrate was the control tube. The test tubes were left in boiling water for 5 minutes, after
which the test tubes were cooled down. Anhydrous sodium carbonate was added to neutralize the
samples, until the samples stopped producing gas or bubbles. Fifteen ml of Fehling’s A and 15.0 ml of
Fehling’s B was mixed to create Fehling’s solution. One ml of Fehling’s solution is then added to both of
the sample tubes. The sample tubes were then placed in a hot water bath for 2 minutes. If the extract
was positive for glycosides, then the hydrolyzed solution produced a red-brown precipitate with a green
suspension.
Test for Saponins (Froth Test) – The extract was dissolved in hot water; after which the solution
was filtered. Two ml of the filtrate in water was placed in a test tube. The test produced a positive result
when a froth was present after shaking the test tube.
Test for Steroids and Triterpenoids (Liebermann-Burchard’s Test) – The dried extract was
treated with a couple of drops of acetic anhydride in a test tube. The solution was then boiled and
allowed to cool. When the solution was cooled, the solution was decanted to leave the solute. One to 2
drops of concentrated sulfuric acid were added by dropping the sulfuric acid down the side of the test
tube. A brown ring was formed in between two formed layers. When the top layer turns green, steroids
were present. When the bottom layer formed a dark red color, then the test showed that triterpenoids
were present. Salkowski’s Test – One to 2 drops of concentrated sulfuric acid were added to the extract.
One to 2 drops of acetic anhydride were also added to the solution in chloroform. The formation of a red
color at the lower layer identified the presence of steroids, whereas if a yellow color was formed, then
there was the presence of triterpenoids.
Test for Alkaloids (Mayer’s Test) – 3.0 ml of the extract is treated with 1.0% HCL and then
filtered. One to 2 drops of Mayer’s reagent were added to the filtrate. A positive result was observed if a
cream-colored filtrate was formed. Wagner’s Test - 1.0 ml of dilute acetic acid is used to treat the sample
extract. One to 2 drops of Wagner’s reagent were dropped onto the extract. A positive result indicated a
brownish or cream-like precipitate.
Test for Flavonoids (Shinoda Test) – The extract was dissolved in ethanol and then filtered. The
filtrate was treated with some drops of 10% concentrated HCl. A positive result of a pink-red color
indicated the presence of flavonoids when magnesium turnings were added to the solution.
Test for Tannins (Ferric Chloride Test)– The extract was cleaned and filtered through ethanol.
One ml of the resulting extract was combined with 2.0 ml water in a sample tube. One to 2 drops of
diluted ferric chloride were placed into the test tube. A positive result of a blue-green or blue-black color
showed the presence of tannins.
Antimicrobial Assays
1. Preparation of Test Organisms Transfer bacterial isolate to 5.0 mL of Tryptic Soy Broth (TSB) in
a test tube. Incubate the tube overnight at 35 °C. After preparation of the bacterial sample, use
for Step B.
2. Preparation of Antimicrobial Assay Plates Adjust the turbidity of the overnight culture with a
solution of sterile saline or broth to 0.5 McFarland standard. Use a sterile non-toxic swab to
transfer the adjusted bacterial suspension onto a petri dish. Add about 15ml-20ml of Mueller-
Hinton Agar, swirl, and allow plates to form and dry, ready for the bacterial swab. Incubate
bacterial plates at 35°C for 30 minutes to an hour depending on the colony. Use filter paper
discs containing samples. Refer to Step C.
3. Addition of Extract/Control Filter Paper Discs
1. Pipette 10uL of the sample into 10mm sterile paper discs, then use sterile forceps to transfer
the discs onto the petri dish containing the colony from Step B. 2)
2. Dip the 10mm paper discs in the sample extract and allow the discs to be marinated by the
sample. Remove excess extracts pressing the discs on the side of a container using sterile
forceps. Transfer to the petri dish in Step B. 1c. For dry extracts and solid samples, take
some of the extract and add 1.0 mL of water. The 10 mm filter paper discs will then be
processed in a similar manner to 1b.
4. Use of Positive Controls
1. Incubation and Interpretation of the Results Incubate the plates inverted overnight at 35°C.
2. Observe for zones of inhibition after incubation.
3. Measure the zones of inhibition using caliper. If there are no zones surrounding the paper
discs, aseptically lift the paper and observe the area under the sample.
RESULTS AND DISCUSSION
The 33-gram black crude extract of Paspalum conjugatum (carabao grass) was examined through
a phytochemical test for its plant constituents with the results of its composition shown below.
Table 1. Phytochemical Test Result
Constituents Results
Sterols (+++)
Triterpenes (++)
Flavonoids (+++)
Alkaloids (+)
Saponins (++)
Glycosides (++)
Tannins (+++)

Note: (+) Traces, (++) Moderate, (+++) Abundant, (-) Absence of constituent
Antimicrobial Assay Test Result The 33g black crude extract of Carabao grass (Paspalum
conjugatum) leaves was put through an antimicrobial assay. The test method was done through disc
diffusion. The extract was set against Staphylococcus aureus with the results of the antimicrobial essay
shown below.
Table 2. Antimicrobial Assay Test

Total mean
zone
Inhibitory
Sample/Control Replicate 1 Replicate 2 Replicate 3 Inhibitory of
Reactivity Activity
inhibition
(mm)
Paspalum
conjugatum
(Carabao 10 10 10 10 2 (+++)
Grass) Extract
(10mm)
Positive
Control:
20.54 - - 20.54 4 (+++)
Oxacillin 1ug
(6mm)
Negative
Control:
0 - - 0 0 (-)
Sample-free
disc (10mm)
Reactivity Rating: 0 - None (No detectable zone around or under specimen)
1 - Slight (Some malformed or degenerated cells under the specimen)
2 - Mild (zone limited under the specimen)
3 - Moderate (zone extends 5 to 10 beyond specimen)
4 - Severe (zone extends greater than 10 mm beyond specimen)
Inhibitory Rating: (+++) complete; (++) partial; (+) slight, and (-) negative

CONCLUSION
After a period of evaluation, the researchers were able to determine the phytochemical
constituents found in carabao grass leaves and its efficacy against Staphylococcus Aureus.
  There were abundant traces of sterols, which have positive effects in the body such as
increased muscle mass, tissue repair, endurance, and fat loss.
 Flavonoids, which have anti-protozoal, anti-inflammatory and antioxidant effects are
beneficial to persons with heart disease and stroke. They can also lower the total cholesterol
found in the blood.
 Tannins have shown anti-diabetic, antimicrobial, anti-Inflammatory, antioxidant and free
radical scavenging activity as well as anti-cancer, antinutritional, cardio-protective properties
and immune-regulating activities.
 There were also a few traces of alkaloids found in the sample known for their action as
analgesics, anesthetics, fibrinolytics, anti-tumor agents, antimalarials, anti-arrhythmic, anti-
bacterial, and as pain killers.
Phytochemical Constituents which are moderately abundant were triterpenes, saponins, and
glycosides. Just like the other phytochemicals these three constituents have their own actions and
benefits, particularly in immune-regulating activities. The results have shown that the carabao grass
leaves extract have complete inhibitory activity with mild reactivity against the test organism
Staphylococcus aureus, which is a bacterium causing skin infections.

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