Preformulation and Evaluation of Antibacterial and Anti-Inflammatory Activity of

Saccharum spontaneum Linn. (Fam. Poaceae) root extract cream

Lapuz, A.M.R., Arabiran, R. D.A., Sembrano, T. M., Albaniel, J. R., Paet, J. C. and Maini, H. A.,
RPh
ABSTRACT
The objective of the present study was to pre-formulate and evaluate the antibacterial and
anti-inflammatory activity of Saccharum spontaneum Linn. root extract cream. The collected
roots of the plant material were air-dried and undergone maceration using 95% ethyl alcohol for
48 hours. Using the Buchner filter, the filtered extract was concentrated under 40C using the
rotary evaporator and then lyophilizer. The 0.5%, 1%, 1.5%, and 2% w/v of concentrated
solution underwent phytochemical screening and the results have identified the presence of
carbohydrates, glycosides, alkaloids, tannins and flavonoids.
Kirby-Bauer Disk Diffusion Method was used to test the antibacterial activity of the root
extract and the pre-formulated cream. The three microorganism used were Staphylococcus
aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa.
The extract with a concentration of 0.5%, 1%, 1.5%, and 2% together with 95% ethanol
were infused in a sterile filter paper and incubated at 37C for 18-24 hours in triplicate. The zone
of inhibition was measured using digital Vernier caliper.
Considering the amount of the ingredients, series of steps were conducted for the preformulation of the cream. At the end, the product was weighed and packed.
The pre-formulated cream was then tested with the same antibacterial screening and then
followed by anti-inflammatory test using selected numbers of mice injected with 0.1 mL of 1%
carrageenan solution in each test groups. The degree of inflammation was measured using a
digital Vernier caliper after at 0, 1, 2, and 3 hours after the stimulus. The antibacterial effect of S.
spontaneum Linn. root extract cream on selected bacteria and its anti-inflammatory activity to
the carrageenan-induced inflammation on Swiss albino mice are therefore discussed. The results
showed that the pre-formulated root extract 2% cream of the plant possess anti-inflammatory
activity rather than antibacterial activity.
Keywords: antibacterial, anti-inflammatory, Kirby-Bauer Disk Diffusion Method, carrageenaninduced paw edema, S. spontaneum Linn., cream
INTRODUCTION
The Philippines, being a tropical country, is a home for diverse species of plants that
strive and grow. Humans are dependent upon these plants. Directly or indirectly, plants provide
food, clothing, fuel, shelter, and many other necessities in life especially medicine.
One of these plants is the Saccharum spontaneum Linn. To some folks in rural areas
where traditional medicine is still being practiced, S. spontaneum Linn. has a significant use. The
decoction of the plant is used as diuretic and root extract is used for the treatment of urinary tract
infection. Sometimes, freshly prepared stems cut into pieces are chewed to relieve stomach pain.
Studies were conducted and revealed that S. spontaneum Linn. contains specific active
constituents which contribute to its effect against certain microbes. Some studies confirm that the

extract has anti-inflammatory effect and can also be used to treat kidney stones (Suresh Kumar,
C.A., 2010)
The researchers intend to use this exceptional characteristic that may develop its use by
formulating a cream which consists of the S. spontaneum Linn. root extract. The researchers
believe that through this evaluation they can confirm its antibacterial and anti-inflammatory
effect.
Objectives
The study aimed to evaluate the formulated S. spontaneum Linn. Root extract cream for
antibacterial and anti-inflammatory properties. Specifically, this study aims to:
1. determine if the S. spontaneum Linn. root extract cream will exert its antibacterial and
anti-inflammatory properties;
2. determine the concentration in which the S. spontaneum Linn. root extract exerts the
antibacterial and anti-inflammatory properties; and
3. compare the stability of formulated S. spontaneum Linn. root extract cream to the
standard cream.
Significance of the Study
The study is significant as it provides a more accessible and readily available alternative
treatment to several patients who have skin disorders.
The formulation and evaluation of topical cream from S. spontaneum Linn. root extract is
relatively cheaper compared to medicines that are already available in the market, more over this
study can contribute in solving economic problem regarding expensive medicine. This is also
another way in helping the less privilege people improve their health condition because it is
affordable.
The study is significant and novel since formulation of any dosage form from S.
spontaneum Linn. extract has never been performed. This study will serve as future reference for
further study on S. spontaneum Linn.
METHODOLOGY
Sample of S. spontaneum Linn. was collected and air dried. The dried plant material was
extracted with 95% ethanol by soaking it for 48 hours with frequent agitation. After extraction, it
was filtered using a Buchner filtration and the filtrate concentrated under reduced pressure at
40C through a rotary evaporator and then lyophilizer. The powdered extract was resuspended in
the solvent to yield 0.5%, 1%, 1.5%, and 2% w/v of solution. The solution then underwent
phytochemical screening to test for the presence of alkaloids, tannins and flavonoids.
Kirby-Bauer disk diffusion method was used to test the antibacterial activity of the S.
spontaneum Linn. root extract and the formulated cream. Three microorganisms, namely, S.
aureus, S. epidermidis, and P. aeruginosa, were used to test the antibacterial activity. All bacteria
that were used in the study were acquired from the Department of Microbiology, College of
Public Health, University of the Philippines Manila.
Sterile filter paper disks was placed and infused with the S. spontaneum Linn. root
extract, standard (Polymyxin B Sulfate + Bacitracin Zinc + Neomycin Sulfate), and negative
control (95% ethanol) on separate plates of prepared culture medium. The plates were incubated
at 37C for 18-24hrs. This was done in triplicate. (Laruan et.al., 2013). After incubation, the zone

of inhibition of the S. spontaneum Linn. root extract, standard and negative controls was
measured and recorded (Laruan et.al., 2013).
The cream was formulated by weighing and measuring the desired amounts of
ingredients. Cetostearyl alcohol was melted in an evaporating dish over a water bath to a
temperature no higher than 60C. After melting, the liquid paraffin was added and then removed
from heat. The mixture was stirred to form the oily phase. The freshly boiled and cooled purified
water to a beaker was transferred and heated to 60C. The S. spontaneum Linn. root extract was
added to the freshly boiled and cooled purified water and then removed from the heat. The
mixture was stirred to form the aqueous phase. When both phases were at about 60C, the
aqueous phase was added to the oily phase with constant, but not too vigorous stirring. The
mixture was stirred until cool enough to pack. Fifty grams of the product was weighed, packed in
a jar and then labeled. (Marriott et.al., 2010).
Table 1. Formulation of the S. spontaneum Linn. root extract cream
Ingredients
Placebo
0.5%
1%
1.5%

2%

Crude ethanolic extract

0g

5.00g

10.00g

15.00g

20.00g

Cetostearyl alcohol

50.25g

49.75g

49.50g

49.25g

Liquid paraffin

502.50g

497.50g

495.00g

492.50g

Distilled water

447.25g

442.75g

440.50g

438.25g

To make

1000g

50.00g
500.00
g
445.00
g
1000g

1000g

1000g

1000g

Test for the properties of cream such as spreadability, washability and pH was employed
on the formulated cream.
Male swiss albino mice weighing 18 25 g were used as test animal for evaluation of
anti-inflammatory activity of the formulated cream. The test animals were procured from the
Department of Pharmacology, College of Medicine, University of the Philippines Manila. They
were maintained in large polypropylene cages and were acclimatized to animal house condition,
fed with commercial pellet and have free access to water.
The Swiss albino mice were divided into six groups having 6 mice in each group. Group I
(control group) received the base cream only; Group II V (test groups) received the formulation
containing different concentration of 0.5%, 1%, 1.5% and 2%, respectively, S. spontaneum Linn.
ethanolic extract by topical administration; Group VI (standard group) received the Diclofenac
sodium 1% gel by topical administration.
The carrageenan-induced paw edema test was used to evaluate the anti-inflammatory
activity of the formulation. The animals pretreated with the formulation or diclofenac sodium
one hour before were injected with 0.1 mL of 1% carrageenan solution into the plant or tissue of
the right hind paw of each mouse. Measure the initial paw volume immediately after carrageenan
application at 0, 1, 2, 3, and 4 hours after the stimulus (Swathy, Mohana Iakshmi, and Saravana
Kumar, 2010).
Statistical Analysis
The data was presented as mean standard deviation of parallel measurements. The
difference between the treatment groups was analyzed using One-way Analysis of Variance

(ANOVA) followed by Dunnetts test using SPSS version 18 software. A p-value of less than
0.05 is considered significant (Swathy et.al., 2010).
RESULTS AND DISCUSSION
Table 2 presented the phytochemical profile of the plant extract of S. spontaneum Linn.
which gave positive results to carbohydrates, glycosides, alkaloids, condensed tannins, and
flavonoids.
Table 2. Phytochemical Screening for S. spontaneum Linn. ethanolic root extract
Test Conducted
Theoretical Result
Actual Result
Test for Carbohydrates
Reddish-violet zone at the Purple ring at the junction
Molischs Test
point of contact
of 2 liquids
Test for Glycosides
Brick-red precipitate in
Fehlings Test
Brick-red precipitate
green solution
Test for Alkaloids
Orange precipitate in
Dragendorffs Test
Orange-brown precipitate
brown solution
Test for Tannins
Blue-black color or
Ferric Chloride Test
Greenish-black solution
brownish-green color
Test for Flavonoids
Bate-Smith-Metcalf Test
Reddish color
Reddish coloration
Table 3 presented the result of the zone of inhibition of the crude extract on the organisms
at 0.5%, 1.0%, 1.5% and 2.0%. At 1.5% and 2.0%, the ethanolic root extract of S.spontaneum
Linn. exerted an antibacterial activity on the two microorganisms, S. aureus and S. epidermidis.
It can be observed that the zone of inhibition gradually increases as the concentration of the
extract increases indicating an increase in antibacterial activity. The zone of inhibition was not
detected in P. aeruginosa which indicates that it has lower or insusceptible to the antibacterial
action of S. spontaneum Linn. root extract. Also, the zone of inhibition was greater for the
positive control as compared to the plant extract; hence, the microorganisms has higher
susceptibility to the antibacterial activity of the positive control than the plant extract.
Table 4 presented the result of the zone of inhibition of the formulated cream on the
microorganisms at 0.5%, 1.0%, 1.5% and 2.0%. The formulated cream did not exhibit any zone
of inhibition at any given concentration; hence, it did not exert any antibacterial action on the
microorganisms used.

Table 5 shows the anti-inflammatory activity of the formulated S. spontaneum Linn. root
extract cream at 0.5% (Group II), 1.0% (Group III), 1.5% (Group IV), and 2.0% (Group V)
concentration, positive control (Group VI) and the negative control (Group I). It was observed
that 1 hour after the application of the sample the inflammation still tends to increase.
At 2 hours after the application the inflammation decreases for the Groups II-VI, except
for Group I (placebo) which indicates no anti-inflammatory activity. It was also observed that the
difference in the decrease of volume was higher for the positive control group which indicates
higher anti-inflammatory activity.
At 3 hours after the application the difference in the decrease in inflammation was higher
for the group V with 2% extract than the other treatment groups including the positive control
group which shows higher anti-inflammatory activity.
Table 5. Difference in the Volume of Inflammation at 1 hour, 2 hours, and 3 hours
after the application of formulated Saccharum spontaneum Linn. root extract
cream and positive control
Treatm
ent
Group
GROUP
1
GROUP
2
GROUP
3
GROUP

After 1
Hour

After 2
hours

After 3
hours

-0.74 0.26

-0.12 0.11

-0.87 0.13

-0.23
0.07
0.33 0.11

-0.52 0.14

0.65 0.16

0.19 0.08

-0.83 0.12

0.82 0.16

0.22 0.08

0.12 0.03

4
GROUP -0.75 0.08
5
GROUP -0.68 0.18
6

1.08 0.15

0.47 0.04

1.68 0.33

0.43 0.12

CONCLUSION
The results obtained showed that the formulated cream has the potential for antiinflammatory activity rather than antibacterial property. The 2% concentration exhibited highest
anti-inflammatory effect. The Kirby-Bauer Disk Diffusion Method showed that there was a
significant difference in the zone of inhibition but lower than of the standard cream (Polymyxin
B Sulfate + Bacitracin Zinc + Neomycin Sulfate).
RECOMMENDATION
The researchers recommend the use of other gram positive and gram negative bacteria,
ready-made agar plates and comparison standard table for more accurate results. Skin sensitivity
test is recommended to ensure that the preformulation would not produce allergic reaction and to
use higher concentrations of the preformulation for future research. The use of this research as
reference is highly appreciated by the researchers.
REFERENCES
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July 2013]
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(L.) Pers. and its ethnomedical importance to indigenous communities of Benguet
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Marriott, J.F., Wilson, K.A., Langley, C.A., and Belcher, D. (2010). Pharmaceutical
Compounding and Dispensing. 2nd Edition. London: Pharmaceutical Press. p.153-157.
Omar, O. (1991). The Pharmacological and Toxicological Studies of Saccharum spontaneum
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spontaneum. International Journal of PharmTech Research. 2(1). 319-321.
Swathy, B., Mohana Iakshimi, S., and Saravana Kumar, A. (2010). Evaluation of Analgesic and
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