Buya Anti-Quorumsensing Rosemary Sage Journal 2016

ANTI-QUORUM SENSING ACTIVITY OF ROSEMARY (Rosmarinus officinalis) AND
SAGE (Salvia officinalis) LEAF EXTRACTS AGAINST METHICILLIN-RESISTANT

Staphylococcus aureus AND Pseudomonas aeruginosa
A Research
Submitted to the Faculty of the
School of Natural Sciences
Saint Louis University
In Partial Fulfillment of the Requirements

for the Degree,
Bachelor in Medical Laboratory Science
Buya, Jennifer T.
Asistin, Ralph Justine A.
Chiok, Jerelyne T.
Feliciano, Reynold G.
Navarro, Angelica Mae B.
Reyes, Edgar Jr. S.
Terrado, Melvin Jethro E.
Venterez, Geena Lindsay F.
May 2016
1
Certificate of Originality
This manuscript hereby submitted by the undersigned researchers serves to present the
results of a study that the undersigned researchers personally conducted. To the best of our
knowledge and belief it has no material previously published or written by another person that is
not properly documented, or permission for its inclusion and use not sought, when necessary.
We certify further that a copy of the PlagScan evaluation is attached to this manuscript (see
attached)
The discourse is my own intellectual exercise, although assistance was received from
others on style, phraseology, presentation, and organization.
S1: ASISTIN, RALPH JUSTINE A.
S5: REYES, EDGAR JR. S.
(signature over printed name)
S2: CHIOK, JERELYNE T.
S6: TERRADO, MELVIN JETHRO E.
S3: FELICIANO, REYNOLD G.

name)
S7: VENTEREZ, GEENA LINDSAY F.
S4: NAVARRO, ANGELICA MAE B.
(signature over printed
S8: ______________________________
Date: June 9, 2016
JENNIFER T. BUYA RMT

FACULTY RESEARCH PROMOTER
Date: June 9, 2016
ENDORSEMENT
In partial fulfillment of the requirements for the degree,

Bachelor in Medical Laboratory Science,
this research entitled,
ANTI- QUORUM SENSING ACTIVITY OF ROSEMARY (Rosmarinus officinalis) AND

prepared and submitted by:


name)
S8: ______________________________
is hereby endorsed for evaluation and approval.

Faculty Research Promoter
Date: June 9, 2016
APPROVAL SHEET
This is to certify that this Research entitled,
ANTI- QUORUM SENSING ACTIVITY OF ROSEMARY (Rosmarinus officinalis) AND
prepared and submitted by


name)
S8: ______________________________
has been approved and accepted as partial fulfillment of the requirements for the degree
Bachelor in Medical Laboratory Science on _______________________.

Faculty Research Promoter
PANEL MEMBERS
RACQUEL C. BARCELO PhD
Member
SANDRA LYN D. DELA PEA

Member
Allan Jay C. Espiritu, RMT, M.Sc.

Undergraduate Research Coordinator
1
Introduction
Centers for Disease Control and Prevention (CDC, 2011) studies show that about one in
three (33%) people carry Staphylococcus aureus in their nose, usually without any illness. Two
in one hundred people carry Methicillin-resistant S. aureus (MRSA).Immunocompromised
patients may acquire the bacteria which may lead to more serious illnesses.
Increase in morbidity, mortality and healthcare costs are reported due to multidrug resistant
organism (MDRO) acquired infections. MRSA is known for its persistence in clinical settings
due to increasing resistance to antimicrobial agents, making it the major pathogen for nosocomial
infection. Biofilm formation appears as its survival strategy(Ando, Monden, Mitsuhata,
Kariyama & Kumon, 2004; Tay & Yew, 2013).
The development of new antibacterial drugs for the treatment of MRSA infections is of
increasing interest (Schito, 2006). The resistance has been emerging not only to methicillin but
also to other many antibiotics, including vancomycin leading to further restriction on available
antibiotic options (Bakri, Al-Hommos, Shehabi, Naffa, Cui & Hiramitsu, 2007; Mohammad,
2010).
Another arising resistant pathogen is Pseudomonas aeruginosa. Strains with resistance are
due to biofilm formation which protects them from antibiotics. In addition, it uses a large arsenal
of pathogenicity factors to interfere with host defenses (Hauser & Ozer, 2011).
Study conducted by Mendoza and So (2015) in the Philippines showed that the most
common antibiotics used were cephalosporin, beta-lactam/beta-lactamase inhibitors then
fluoroquinolones. Antibiotics such as piperacillin-tazobactam, ceftriaxonolones and tetrapenem
increased significantly decreasing the prevalence of P. aeruginosa while MRSA remains
unchanged.
Bacteria have certain mechanisms which allow them to survive. One is called quorum
sensing. Quorum sensing (QS) is a signalling phenomenon used by bacteria in order to express
virulence (Vattem, Mihalik, Crixell & McLean, 2007). Mechanisms of QS include: gene
expression, virulence expression, biofilm formation, sporulation, bioluminescence, and mating
(Nazarro, Fratianni & Coppola, 2013). Koh, Sam, Yin, Tan, Krishnan, Chong and Chan (2013)
defined it as a system of stimuli and responses in relation to bacterial cell population density that
regulates gene expression, including virulence determinants.
Since several processes responsible for successful establishment of bacterial infection are
mediated by quorum sensing (Vattem et al., 2007), it has been an attractive target for the
development of novel anti-infective measures that do not rely on the use of antibiotics (Koh et
al., 2013).
QS is the communication mechanism between bacterial cells that enables them to control the
way genes are expressed. This type of communication of microorganisms is only achieved at
higher cell densities. The bacteria release autoinducers, various molecules that act as mediators
of quorum sensing (Raina, De Vizio, Odell, Clements & Vanhulle, 2009). The molecules that are
implicated in quorum sensing should satisfy four essential characteristics. First, a quorum
sensing molecule must be able to regulate its own production. Second, is that it should be
produced throughout the growth of the organism since it is dependent to cell density. Third, it
must be able to elicit a similar response in a mutant strain that has a deficiency in producing the
autoinducers with the exogenous addition of the quorum sensing molecule. Lastly, a quorum
sensing molecule must be able to respond not only related to metabolizing or detoxifying the
molecule itself. It should respond physiologically in the organism in which it is produced (Raina
et al., 2009).
2
The quorum sensing molecules can be degraded or inhibited in order to disrupt quorum
sensing.
Quorum sensing antagonists are being investigated in targeting the signal receptor
(Raina et al., 2009).
Two other mechanisms were suggested by basic researchers aside from targeting signal
generation, dissemination, and reception. One example is to use the quorum sensing molecules
not to kill infectious bacteria but to have an effect in the modification of the behaviour such as
turning off of virulence (Hentzer, Riedel, Rasmussen, Heydorn & Andersen, 2003).
Both P. aeruginosa and MRSA exhibit quorum sensing activities. These make them
appropriate test organisms in searching for compounds that could inhibit quorum sensing
activity.
World Health Organization encouraged health systems in different countries since 1980s
to interact with herbal medicine for identifying and assessing means that build up bases for new
and safe herbal agents which can be used for treatment of infectious and noninfectious diseases
(WHO, 1978).
Certain bacteria can become resistant to certain antibiotics when surviving strains are left to
reproduce (CDC, 2011). The increasing amounts of antibiotics developed over the past 60 years
have provoked bacteria to respond by propagating progeny no longer susceptible to these
antibiotics making the development of newer generations of antibiotics increasingly obsolete as a
method of combating pathogenic bacteria (Levy, 2000). Drugs that disrupt quorum sensing, on
the other hand, would spare the microorganism, simply preventing them from causing disease or
causing antibiotic resistance (Wenner, 2009).
Several plants and natural products were initially examined for their traditional medicine use
and recent studies suggest that these can also be a source of natural inhibitors for QS activity
(Koh et al., 2013; Zahin, Hasan, Aqil, Khan, Husain & Ahmad, 2010; and Tolmacheva,
Rogozhin, & Deryabin 2014). According to Szab et al. (2010), rosemary oil is a potent quorum
sensing inhibitor against Chromobacterium violaceum. Another study by Nagy (2010) showed
several plant extracts including rosemary exhibiting anti- quorum sensing activity against C.
violaceum and P. aeruginosa. Thus rosemary can be a potent source for anti-quorum sensing
activity of microorganism. Sage (Salvia officinalis) is of the same family as that of rosemary
(Rosmarinus officinalis) which is the Labiatae family. According to researches done by
Hippolyte, Marin, Baccou & Jonard (1992), a natural antioxidant called rosmarinic acid is
produced by the cell suspension cultures of sage. With the same chemical component utilized in
a study conducted by Koh et al. (2013) and Nagy (2010), rosmarinic acid extracted from sweet
basil decreased the expression of the elastase and protease, as well as biofilm formation in P.
aeruginosa.
Assessment of the anti-quorum sensing properties of these plant extracts, which are locally
available, offers an alternative to the reduction of antibiotic resistance development. Specific
problems addressed by the study include: first the determination of significant difference in
biofilm formation between the extract-treated and untreated cultures of P. aeroginosa with
different extract concentrations; determination of significant difference in biofilm formation
between extract-treated and untreated cultures of mecA-negative S. aureus with different extract
concentrations and determination of the significant difference in antibiotic resistance between
extract-treated and untreated cultures of mecA-positive S. aureus as measured by quantity of
bacterial cells as expressed through optical density and zone of inhibition in the cefoxitin
diffusion method.
3
Research method and design
This research utilized the experimental method of research following the factorial
experimental design. The independent variables are the presence/absence of the anti-quorum
sensing compounds that would be extracted from Rosemary (R. officinalis) and Sage (S.
officinalis), and the concentrations of these extracts in the treatment groups. The dependent
variables would be the biosensor indicators identified for each assay.
Anti-quorum Sensing Compound Extract Preparation
Mature leaves of Rosemary and Sage were collected from Alapang, La Trinidad, Benguet.
Extraction was done by initially washing the collected leaves, sun drying them and finally
placing them in the oven to eliminate their moisture content. The oven processed leaves were
then soaked in alcohol (95% ethanol) for 48 hours. Concentration process was done thru Soxhlet
method for 4 hours or more to obtain the leaf extract. The crude extract was placed in a water
bath to remove the excess alcohol to obtain a crude extract. It was then dissolved with dimethyl
sulfoxide (DMSO) to produce a stock solution having a concentration of 100 g/mL.
Different concentrations of extracts were prepared from the stock solution and placed in an
amber bottle then autoclaved at 120C to remove impurities. The concentrations prepared were as
follows: 10 g/mL, 20 g/mL, 30 g/mL, 40 g/mL, 50 g/mL, 60 g/mL, 70 g/mL, 80
g/mL, 90 g/mL and 100 g/mL. Additional concentration of extract at 1000 g/mL was also
prepared for the antibiotic resistance assay.
Biofilm Inhibition Assay using P. aeruginosa PA01 and/or Methicillin-resistant S. aureus
This assay was adapted from Ando et al. (2004). P. aeruginosa PA01 was grown overnight at
o
37 C in a nutrient broth. The culture was diluted 1:100 in medium, and 50 L of the cell
suspension was inoculated into sterile 96-well polystyrene microtiter plates previously added
with 50 L of 10-fold increasing concentrations of the extracts from 0% v/v to 100% v/v diluted
with distilled water. The plates were incubated for 48 hours at 37 oC without shaking. After
incubation, the wells were gently washed three times with 200 L of distilled water, dried in an
inverted position, and stained with 200 L of 2% crystal violet solution in water for 45 minutes.
The wells were destained with 200 L ethanol-acetic acid (95:5, v/v). Two hundred microliters
from each well was transferred into a cuvette containing 800 L of distilled water, and the optical
density of crystal violet present in the destaining solution was measured at 545-580 nm using a
spectrophotometer. The same procedures were carried out with MRSA.
As a control, an uninoculated medium was used to determine background OD. It was
used to set the spectrophotometer at zero absorbance reading. A negative control containing
tripticase soy broth was prepared which has the lowest absorbance reading. A positive control
containing a bacterial suspension (P. aeruginosa and MRSA) was also prepared and has the
highest absorbance reading.
The presence of anti-quorum sensing activity (biofilm formation inhibition) is a shown by
a decrease in absorbance readings of the solutions with extract when compared to the positive
control.
4
Antibiotic Resistance Assay in mecA(+) Methicillin-Resistant S. aureus
Quantitative tube method
A culture tube containing nutrient broth was inoculated with mecA-positive methicillinresistant S. aureus (MRSA) ATCC 43300 and incubated at 37oC for six hours with 100 L
supplementation with sterile test extract every two hours. Another tube was also inoculated and
incubated following the same conditions as the first but without supplementation. One hundred
microliters of direct culture suspensions equivalent to 0.5 McFarland standard from the two tubes
was separately sub-cultured onto a) Tryptic Soy Broth culture tube with 4% NaCl and 6g/mL
oxacillin, and, b) Tryptic Soy Broth culture tube without supplements. The four resulting tubes
were incubated at 37oC for 16-18 hours and growth was assessed spectrophotometrically at 570
nm.
mecA-mediated Oxacillin Resistance using Cefoxitin (Disk Diffusion Method)
A culture tube containing nutrient broth was inoculated with mecA-positive methicillinresistant S. aureus (MRSA) ATCC 43300 and was incubated at 37 oC for six hours with 100 L
supplementation with sterile test extract every two hours. Another tube was also inoculated and
incubated following the same conditions as the first but without supplementation. Direct colony
suspensions equivalent to 0.5 McFarland standard from the two tubes were separately plated onto
Mueller Hinton Agar plate with 4% NaCl and 6g/mL oxacillin then three 30 g cefoxitin disks
were introduced equidistantly in each plate. The two resulting plates were incubated at 37 oC for
16-18 hours.
Treatment of Data
All measured values in each assay were subjected to Analysis of Variance to determine
significant differences. Statistically significant sets were further analyzed using Tukeys HSD to
determine where the significant differences lie. Confidence levels were uniformly set at 0.05
alpha.
Results and Discussion
Biofilm Inhibition Assay using P. aeruginosa PA01
Screening of anti-quorum sensing activity of extracts of rosemary and sage was done by
using P. aeruginosa as biosensor. The treatment includes 10 different concentrations of the
extract, tripticase soy broth as the negative control, and P. aeruginosa overnight culture as the
positive control.
Table no. 1 Tests of Between-Subjects Effects
Concentrations
Groups
Concentrations*Group
s
.000
.112
Rosemary extract
.000
.000
Sage extract
.000
.001
Lesser than 0.05 indicates significance
The concentrations refer to the 10 different concentrations of extract used. The groups
refer to the comparison between group 1 and 2. Group 1 contained the extract and
microorganism while group 2 contained the extract only.
5
Rosemary extract shows significant interaction of the different concentrations which means
that the mean absorbances are statistically different as affected by the different concentrations.
The mean absorbance of the groups shows significant difference, which means that with the
microorganism, biofilm is produced and without the microorganism, no biofilm is produced. This
shows that the microorganism was not killed entirely, corresponding to its anti-quorum sensing
activity rather than antibiotic activity. The interaction between concentration and group show
significant difference which means that the different concentration of the extract used to treat the
groups, significant decrease in biofilm is observed.
Sage extract shows significant interaction of the different concentrations which means that
the mean absorbances are statistically different as affected by the different concentrations. The
mean absorbance of the groups shows significant difference, which means that with or without
the microorganism, there is no biofilm produced. This shows that the microorganism was killed,
corresponding to its antibiotic activity rather than anti-quorum sensing activity. The interaction
between concentration and group show no significant difference which means that regardless of
the concentration of the extract used to treat the groups, no significant decrease in biofilm is
observed.
Table no.2 Means for groups in Homogenous Subsets for Rosemary
Tukey HSDa,b,c
Concentrations
N
Subset
1
2
3
1
6
.04367
11
6
.05883
12
6
.06550
Sig.
.195
1.000
.882
Same subset indicates no significant difference
Concentrations 1 and 12 are significant. This means that the microorganism decreased its
biofilm formation at the lowest concentration of the extracts. Concentration 12 (positive control)
is significantly different from the other wells. Concentrations 2 to 10 show no significant
difference, which means that biofilm formation is reduced regardless of the different
concentrations.
Table no.3 Means for groups in Homogenous Subsets for Sage
Concentration
N
Subset
s
1
2
3
11
6
.03533
.03533
1
5
.04700
.04700
12
6
.06383
Sig.
.310
.074
.637
Concentrations 1 and 12 are not significant. This means that the biofilm formation at the
lowest concentration of the extract was not significantly reduced. Concentrations 11 (negative
control) and 12 (positive control) have a significant difference, which means that the positive
6
control produced biofilm while the negative control did not. Concentrations 2 to 11 show no
significant difference, this means that biofilm formation is reduced regardless of the different
concentrations of the extracts.
Biofilm Inhibition Assay using S. aureus
Second screening of anti-quorum sensing properties of extracts of rosemary and sage was
done by using S. aureus as biosensor. The treatment includes 10 different concentrations of the
extract, tripticase soy broth as the negative control, and S. aureus overnight culture as the
positive control.
Table no. 4 Tests of Between-Subjects Effects
Concentrations
Groups
Concentrations
*Groups
.946
.818
Rosemary extract
.122
.854
Sage extract
.094
.991
Lesser than 0.05 indicates significance
The concentrations refer to the 10 different concentrations of extract used. The groups refer
to the comparison between group 1 and 2. Group 1 contained the extract and microorganism
while group 2 contained the extract only.
Rosemary extract shows no significant interaction of the different concentrations which
means that the mean absorbances are not statistically different as affected by the different
concentrations. The mean absorbance of the groups shows no significant difference, which
means that with or without the microorganism, there is no biofilm produced. This shows that the
microorganism was killed, corresponding to its antibiotic activity rather than anti quorum sensing
activity. The interaction between concentration and group show no significant difference which
means that regardless of the concentration of the extract used to treat the groups, no significant
decrease in biofilm is observed.
Sage extract shows no significant interaction of the different concentrations which means
that the mean absorbances are not statistically different as affected by the different
concentrations. The mean absorbance of the groups shows no significant difference, which
means that with or without the microorganism, there is no biofilm produced. This shows that the
microorganism was killed, corresponding to its antibiotic activity rather than anti quorum sensing
activity. The interaction between concentration and group show no significant difference which
means that regardless of the concentration of the extract used to treat the groups, no significant
decrease in biofilm is observed.
Table no.5 Means for groups in Homogenous Subsets for Rosemary
Tukey HSDa,b,c
Subset
Concentrations
N
1
1
6
.04383
11
6
.06300
12
6
.15867
Sig.
.085
The 10 concentrations do not differ significantly. This means that the use of the different
extract concentrations do not significantly reduce biofilm formation.
Table no.6 Means for groups in Homogenous Subsets for Sage
Tukey HSDa,b
Subset
Concentrations
N
1
1
6
.04383
11
6
.05467
12
6
.16117
Sig.
.059
The 10 concentrations do not differ significantly. This means that the use of the different
extracts do not significantly reduce biofilm formation. The results show that concentration 1 of
the test shows anti-quorum sensing activity while concentrations 2 to 10 show antibiotic
property. According to Koh et al. (2013), rosemary exhibited anti-quorum sensing activity on P.
aeruginosa and S. aureus although concentrations were not determined. According to Szabo and
Hohmann (2010), rosemary essential oils exhibit potent anti-quorum sensing activity on C.
violaceum, which is also one of the bacterium used as biosensor in determining anti-quorum
sensing activity of plants.
Antibiotic resistance assay
Anti-quorum sensing activity against MRSA was evaluated using the quantitative tube
method and mecA-mediated oxacillin resistance using cefoxitin (disk diffusion method).Positive
and negative controls controls were used to evaluate the results. RNA III Inhibiting Peptide
(RIP), a heptapeptide that inhibits S. aureus pathogenesis by disrupting quorum-sensing
mechanisms, was used as a supplement for positive control (Balaban, Giacometti, Cirioni, Gov,
Ghiselli, Mocchegiani, Viticchi, Del Prete, Saba, Scalise and DellAcqua, 2002).
Quantitative tube method
The test of between subjects of replicates show that the extract for rosemary shows a
0.000 significance while for sage there is 0.069 significance for concentration and 0.017 for
concentration*supplement.
Table no. 7 Antibiotic resistance assay: Quantitative tube method
Volume and
Sage
Rosemary
Medium
Concentration
Extract
Extract
6 mL of 100 L/mL
0.471333
0.662667
6 mL of 10 L/mL
0.587000
0.622000
TSB
600 L of 1000
0.745333
0.724000
L/mL
600 L of 100 L/mL 0.811667
0.500000
TSB
+ 6 mL of 100 L/mL
0.056333
0.016000
Oxacillin 6 mL of 10 L/mL
0.095000
0.161000
Positive
Control
0.587333
0.517333
Negative
Control
0.77200
0.77200
0.741333
0.77200
0.733333
0.183667
0.613667
0.77200
0.662667
0.662667
8
600 L of 1000
L/mL
600 L of 100 L/mL
0.074333
0.033333
0.055000
0.662667
0.026000
0.003666
0.062333
0.662667
This table shows that from the initial growth in the TSB medium, there are decreasing
volumes and concentrations in both solutions. This may prove the possibility that the
microorganism is killed by Oxacillin. Thus, antibiotic activity may indirectly decrease the
growth observed. Similar observations were noted by Nagy (2010) on the activity of the both
extract on C. voilaceum.
mecA-mediated Oxacillin Resistance using Cefoxitin (Disk Diffusion Method)
Table no.8 Mean Diameter of the Zone of Inhibition in mm of the Replicates
Volume and
Sage
Rosemary
Positive
Medium
Concentration
Extract
Extract
Control
6 mL of 100 g/mL
20.3333
20.0000
12
6 mL of 10 g/mL
19.3333
18.0000
11
600 mL of 1000
TSB
18.6670
18.6670
18
g/mL
600 mL of 100
18.0000
22.0000
20
g/mL
Negative
Control
19
19
19
19
The 100 g/mL supplemented in 6 mL shows to have the highest diameter of the different
volume and concentration of sage and the 600 mL of 100 g/mL shows to be the highest in
rosemary. For the 6 mL of 10 g/mL and 600 mL of 1000 g/mL of the sage extract shows to
have an inhibition of 19. These concentrations are believed to have anti-quorum sensing activity
of the extract.
A value of 18 diameters in 600 mL of 100 g/mL in sage extract and 6 mL of 10 g/mL of
rosemary showed to be the lowest. For the 6 mL of 10 g/mL and 600 mL of 1000 g/mL of the
sage extract have an inhibition of 19 and those 6 mL of 100 g/mL and 600 mL of 1000 g/mL
of the rosemary extract showed 20 and 18.6670, respectively. These concentrations do not show
anti-quorum sensing activity instead it can be interpreted as antibacterial. Antibacterial activity
of sage and rosemary was also observed by Alzoubi, Ibrahim, Alsbou, and Aqel (2014).
According to Tolmacheva et al. (2014), sage has antimicrobial activity against C. violaceum.
The activity of the two extracts is more of antibiotic rather than anti-quorum sensing.
However, when drugs are used so widely and for so long, the antibiotics designed to kill
infectious microorganisms have adapted to them, making the drugs less effective (CDC, 2016).
Conclusion and Recommendation
The researchers therefore conclude that R. officinalis leaf extract has anti-quorum sensing
activity against P. aeruginosa and S. aureus at the concentration of 10g/mL as exemplified in
the inhibition of biofilm formation. Concentrations greater than 10g/mL have antibiotic activity.
S. officinalis leaf extract has no anti-quorum sensing activity against P. aeruginosa and S.
aureus at the concentration of 10g/mL, antibiotic activity was rather observed.
In the antibiotic resistance assays, all volumes and concentrations of the rosemary and
sage extract preparations exhibited antibiotic properties.
9
It can therefore be said that the antibiotic and anti-quorum sensing activity of rosemary and
sage extracts is due to independent activity of its components.
The researchers recommend the study of lower concentrations of both rosemary and sage in
order to determine the minimum concentration of the solutions which allow them to exhibit antiquorum sensing activity. Isolation and evaluation for anti-quorum sensing activity of the
rosmarinic acid component will also make the study more specific.
10
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Schito, G.C. (2006). The importance of the development of antibiotic resistance in
Staphylococcus aureus. Retrieved from: http://www.ncbi.nlm.nih.gov/pubmed/16445718
Szab MA, Varga GZ, Hohmann J. (2010). Inhibition of quorum-sensing signals by essential
oils. Phytotherapy Research 24:782-786. http://www.ncbi.nlm.nih.gov/pubmed/19827025
Tay S.B. and Yew W.S. (2013). Development of quorum-based anti-virulence therapeutics
targeting
Gram-negative
bacterial
pathogens.
Retrieved
from:
http://www.ncbi.nlm.nih.gov/pubmed/23939429
Tolmacheva, A. A., Rogozhin, E. A., & Deryabin, D. G. (2014). Antibacterial and quorum
sensing regulatory activities of some traditional eastern-european medicinal plants. Acta
pharm, 64, 173-186. http://dx.doi:10.2478/acph-2014-0019
Vattem, D. A., Mihalik, K., Crixell, S. H., & McLean R.J.C. (2007). Dietary phytochemicals as
quorum
sensing
inhibitors.
Fitoterapia,
78,
302-310.
http://dx.doi:10.1016/j.fitote.2007.03.009
Wenner M. (2009). Quiet Bacteria and Antibiotic resistance. Retrieved from:
http://www.scientificamerican.com/article/bacteria-antibiotic-resistance/
World Health Organization (WHO), author. 1978. The promotion and development of traditional
medicine. Geneva: WHO; Technical Report Series. P: 622
Zahin, M., Hasan, S., Aqil, F., Khan M.S.A., Husain, F.M., and Ahmad, I. (2010). Screening of
certain medical plans from India for their anti-quorum sensing activity. Retrieved from:
http://nopr.niscair.res.in/bitstream/123456789/10652/1/IJEB%2048(12)%201219-1224.pdf
12
APPENDIX A: COMPLETE DATA
BIOFILM FORMATION ASSAY USING Pseudomonas aeruginosa
ROSEMARY
1. Tests of Between-Subjects Effects
Dependent Variable: Absorbances
Type III Sum of
Source
Squares
df
Mean Square
a
Corrected Model
.029
23
.001
Intercept
.060
1
.060
Concentrations
.020
11
.002
Group
.004
1
.004
Concentrations * Group
.006
11
.001
Error
.002
48
4.910E-5
Total
.091
72
Corrected Total
.031
71
F
25.732
1219.173
36.745
72.429
10.473
Sig.
.000
.000
.000
.000
.000
a. R Squared = .925 (Adjusted R Squared = .889)

2. Multiple Comparison
Tukey HSD
(I) Concentrations (J) Concentrations

1
2
3
4
5
6
7
8
9
10
11
12
2
1
3
4
5
6
7
8
9
10
11
12
3
1
2
4
5
6
Mean
Difference (IJ)
Std. Error
.01883*
.004045
*
.02267
.004045
.02050*
.004045
.02233*
.004045
*
.02350
.004045
.02317*
.004045
.02533*
.004045
*
.03033
.004045
.02833*
.004045
*
-.01517
.004045
-.02183*
.004045
-.01883*
.004045
.00383
.004045
.00167
.004045
.00350
.004045
.00467
.004045
.00433
.004045
.00650
.004045
.01150
.004045
.00950
.004045
-.03400*
.004045
-.04067*
.004045
-.02267*
.004045
-.00383
.004045
-.00217
.004045
-.00033
.004045
.00083
.004045
95% Confidence Interval

Sig.
Lower Bound Upper Bound
.001
.00494
.03272
.000
.00878
.03656
.000
.00661
.03439
.000
.00844
.03622
.000
.00961
.03739
.000
.00928
.03706
.000
.01144
.03922
.000
.01644
.04422
.000
.01444
.04222
.022
-.02906
-.00128
.000
-.03572
-.00794
.001
-.03272
-.00494
.998
-.01006
.01772
1.000
-.01222
.01556
.999
-.01039
.01739
.990
-.00922
.01856
.995
-.00956
.01822
.898
-.00739
.02039
.195
-.00239
.02539
.459
-.00439
.02339
.000
-.04789
-.02011
.000
-.05456
-.02678
.000
-.03656
-.00878
.998
-.01772
.01006
1.000
-.01606
.01172
1.000
-.01422
.01356
1.000
-.01306
.01472
13
7
8
9
10
11
12
1
2
3
5
6
7
8
9
10
11
12
1
2
3
4
6
7
8
9
10
11
12
1
2
3
4
5
7
8
9
10
11
12
1
2
3
4
5
6
8
9
10
11
12
1
.00050
.00267
.00767
.00567
-.03783*
-.04450*
-.02050*
-.00167
.00217
.00183
.00300
.00267
.00483
.00983
.00783
-.03567*
-.04233*
-.02233*
-.00350
.00033
-.00183
.00117
.00083
.00300
.00800
.00600
-.03750*
-.04417*
-.02350*
-.00467
-.00083
-.00300
-.00117
-.00033
.00183
.00683
.00483
-.03867*
-.04533*
-.02317*
-.00433
-.00050
-.00267
-.00083
.00033
.00217
.00717
.00517
-.03833*
-.04500*
-.02533*
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
1.000
1.000
.756
.958
.000
.000
.000
1.000
1.000
1.000
1.000
1.000
.987
.406
.731
.000
.000
.000
.999
1.000
1.000
1.000
1.000
1.000
.705
.938
.000
.000
.000
.990
1.000
1.000
1.000
1.000
1.000
.864
.987
.000
.000
.000
.995
1.000
1.000
1.000
1.000
1.000
.825
.978
.000
.000
.000
-.01339
-.01122
-.00622
-.00822
-.05172
-.05839
-.03439
-.01556
-.01172
-.01206
-.01089
-.01122
-.00906
-.00406
-.00606
-.04956
-.05622
-.03622
-.01739
-.01356
-.01572
-.01272
-.01306
-.01089
-.00589
-.00789
-.05139
-.05806
-.03739
-.01856
-.01472
-.01689
-.01506
-.01422
-.01206
-.00706
-.00906
-.05256
-.05922
-.03706
-.01822
-.01439
-.01656
-.01472
-.01356
-.01172
-.00672
-.00872
-.05222
-.05889
-.03922
.01439
.01656
.02156
.01956
-.02394
-.03061
-.00661
.01222
.01606
.01572
.01689
.01656
.01872
.02372
.02172
-.02178
-.02844
-.00844
.01039
.01422
.01206
.01506
.01472
.01689
.02189
.01989
-.02361
-.03028
-.00961
.00922
.01306
.01089
.01272
.01356
.01572
.02072
.01872
-.02478
-.03144
-.00928
.00956
.01339
.01122
.01306
.01422
.01606
.02106
.01906
-.02444
-.03111
-.01144
14
10
11
12
2
3
4
5
6
7
9
10
11
12
1
2
3
4
5
6
7
8
10
11
12
1
2
3
4
5
6
7
8
9
11
12
1
2
3
4
5
6
7
8
9
10
12
1
2
3
4
5
6
7
8
-.00650
-.00267
-.00483
-.00300
-.00183
-.00217
.00500
.00300
-.04050*
-.04717*
-.03033*
-.01150
-.00767
-.00983
-.00800
-.00683
-.00717
-.00500
-.00200
-.04550*
-.05217*
-.02833*
-.00950
-.00567
-.00783
-.00600
-.00483
-.00517
-.00300
.00200
-.04350*
-.05017*
.01517*
.03400*
.03783*
.03567*
.03750*
.03867*
.03833*
.04050*
.04550*
.04350*
-.00667
.02183*
.04067*
.04450*
.04233*
.04417*
.04533*
.04500*
.04717*
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.004045
.898
1.000
.987
1.000
1.000
1.000
.983
1.000
.000
.000
.000
.195
.756
.406
.705
.864
.825
.983
1.000
.000
.000
.000
.459
.958
.731
.938
.987
.978
1.000
1.000
.000
.000
.022
.000
.000
.000
.000
.000
.000
.000
.000
.000
.882
.000
.000
.000
.000
.000
.000
.000
.000
-.02039
-.01656
-.01872
-.01689
-.01572
-.01606
-.00889
-.01089
-.05439
-.06106
-.04422
-.02539
-.02156
-.02372
-.02189
-.02072
-.02106
-.01889
-.01589
-.05939
-.06606
-.04222
-.02339
-.01956
-.02172
-.01989
-.01872
-.01906
-.01689
-.01189
-.05739
-.06406
.00128
.02011
.02394
.02178
.02361
.02478
.02444
.02661
.03161
.02961
-.02056
.00794
.02678
.03061
.02844
.03028
.03144
.03111
.03328
.00739
.01122
.00906
.01089
.01206
.01172
.01889
.01689
-.02661
-.03328
-.01644
.00239
.00622
.00406
.00589
.00706
.00672
.00889
.01189
-.03161
-.03828
-.01444
.00439
.00822
.00606
.00789
.00906
.00872
.01089
.01589
-.02961
-.03628
.02906
.04789
.05172
.04956
.05139
.05256
.05222
.05439
.05939
.05739
.00722
.03572
.05456
.05839
.05622
.05806
.05922
.05889
.06106
15
9
.05217*
10
.05017*
11
.00667
Based on observed means.
The error term is Mean Square(Error) = 4.910E-5.
*. The mean difference is significant at the .05 level.
.004045
.004045
.004045
.000
.000
.882
.03828
.03628
-.00722
.06606
.06406
.02056
3. Homogeneous subsets
Tukey HSDa,b
Concentrations
9
10
8
6
7
3
5
4
2
1
11
12
Sig.
N
6
6
6
6
6
6
6
6
6
6
6
6
1
.01333
.01533
.01833
.02017
.02050
.02100
.02133
.02317
.02483
Subset
2
.04367
.195
.05883
.06550
.882
1.000
Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 6.000.
b. Alpha = .05.
SAGE
Type III Sum of
Source
Squares
Corrected Model
.020a
Intercept
.054
Concentrations
.014
Group
.002
.004
Error
.009
Total
.084
Corrected Total
.029
df
23
1
11
1
11
47
71
70
Mean Square
.001
.054
.001
.002
.000
.000
F
4.566
280.246
6.554
11.768
1.663
Sig.
.000
.000
.000
.001
.112

5.
Multiple Comparison
Multiple Comparisons

Tukey HSD
Mean
Difference (IJ)

Std. Error
Sig.
16
1
2
3
4
5
6
7
8
9
10
11
12
1
3
4
5
6
7
8
9
10
11
12
1
2
4
5
6
7
8
9
10
11
12
1
2
3
5
6
7
8
9
10
11
12
1
2
3
4
6
7
.01867
.02533
.02333
.02650
.01250
.03033*
.03167*
.03267*
.03200*
.01167
-.01683
-.01867
.00667
.00467
.00783
-.00617
.01167
.01300
.01400
.01333
-.00700
-.03550*
-.02533
-.00667
-.00200
.00117
-.01283
.00500
.00633
.00733
.00667
-.01367
-.04217*
-.02333
-.00467
.00200
.00317
-.01083
.00700
.00833
.00933
.00867
-.01167
-.04017*
-.02650
-.00783
-.00117
-.00317
-.01400
.00383
.008411
.008411
.008411
.008411
.008411
.008411
.008411
.008411
.008411
.008411
.008411
.008411
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008411
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008411
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008411
.008019
.008019
.008019
.008019
.008019
.545
.137
.224
.100
.937
.032
.021
.015
.019
.960
.690
.545
.999
1.000
.998
1.000
.945
.892
.838
.876
.999
.003
.137
.999
1.000
1.000
.900
1.000
1.000
.999
.999
.857
.000
.224
1.000
1.000
1.000
.967
.999
.996
.989
.994
.945
.000
.100
.998
1.000
1.000
.838
1.000
-.01024
-.00358
-.00558
-.00241
-.01641
.00142
.00276
.00376
.00309
-.01724
-.04574
-.04758
-.02090
-.02290
-.01973
-.03373
-.01590
-.01457
-.01357
-.01423
-.03457
-.06307
-.05424
-.03423
-.02957
-.02640
-.04040
-.02257
-.02123
-.02023
-.02090
-.04123
-.06973
-.05224
-.03223
-.02557
-.02440
-.03840
-.02057
-.01923
-.01823
-.01890
-.03923
-.06773
-.05541
-.03540
-.02873
-.03073
-.04157
-.02373
.04758
.05424
.05224
.05541
.04141
.05924
.06058
.06158
.06091
.04058
.01208
.01024
.03423
.03223
.03540
.02140
.03923
.04057
.04157
.04090
.02057
-.00793
.00358
.02090
.02557
.02873
.01473
.03257
.03390
.03490
.03423
.01390
-.01460
.00558
.02290
.02957
.03073
.01673
.03457
.03590
.03690
.03623
.01590
-.01260
.00241
.01973
.02640
.02440
.01357
.03140
17
10
8
9
10
11
12
1
2
3
4
5
7
8
9
10
11
12
1
2
3
4
5
6
8
9
10
11
12
1
2
3
4
5
6
7
9
10
11
12
1
2
3
4
5
6
7
8
10
11
12
1
2
.00517
.00617
.00550
-.01483
-.04333*
-.01250
.00617
.01283
.01083
.01400
.01783
.01917
.02017
.01950
-.00083
-.02933*
-.03033*
-.01167
-.00500
-.00700
-.00383
-.01783
.00133
.00233
.00167
-.01867
-.04717*
-.03167*
-.01300
-.00633
-.00833
-.00517
-.01917
-.00133
.00100
.00033
-.02000
-.04850*
-.03267*
-.01400
-.00733
-.00933
-.00617
-.02017
-.00233
-.00100
-.00067
-.02100
-.04950*
-.03200*
-.01333
.008019
.008019
.008019
.008019
.008019
.008411
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008411
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008411
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008411
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008411
.008019
1.000
1.000
1.000
.782
.000
.937
1.000
.900
.967
.838
.542
.432
.356
.406
1.000
.028
.032
.945
1.000
.999
1.000
.542
1.000
1.000
1.000
.473
.000
.021
.892
1.000
.996
1.000
.432
1.000
1.000
1.000
.369
.000
.015
.838
.999
.989
1.000
.356
1.000
1.000
1.000
.299
.000
.019
.876
-.02240
-.02140
-.02207
-.04240
-.07090
-.04141
-.02140
-.01473
-.01673
-.01357
-.00973
-.00840
-.00740
-.00807
-.02840
-.05690
-.05924
-.03923
-.03257
-.03457
-.03140
-.04540
-.02623
-.02523
-.02590
-.04623
-.07473
-.06058
-.04057
-.03390
-.03590
-.03273
-.04673
-.02890
-.02657
-.02723
-.04757
-.07607
-.06158
-.04157
-.03490
-.03690
-.03373
-.04773
-.02990
-.02857
-.02823
-.04857
-.07707
-.06091
-.04090
.03273
.03373
.03307
.01273
-.01577
.01641
.03373
.04040
.03840
.04157
.04540
.04673
.04773
.04707
.02673
-.00177
-.00142
.01590
.02257
.02057
.02373
.00973
.02890
.02990
.02923
.00890
-.01960
-.00276
.01457
.02123
.01923
.02240
.00840
.02623
.02857
.02790
.00757
-.02093
-.00376
.01357
.02023
.01823
.02140
.00740
.02523
.02657
.02690
.00657
-.02193
-.00309
.01423
18
3
-.00667
4
-.00867
5
-.00550
6
-.01950
7
-.00167
8
-.00033
9
.00067
11
-.02033
12
-.04883*
11
1
-.01167
2
.00700
3
.01367
4
.01167
5
.01483
6
.00083
7
.01867
8
.02000
9
.02100
10
.02033
12
-.02850*
12
1
.01683
2
.03550*
3
.04217*
4
.04017*
5
.04333*
6
.02933*
7
.04717*
8
.04850*
9
.04950*
10
.04883*
11
.02850*
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008411
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008411
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
.008019
Absorbances
Tukey HSDa,b,c
Concentrations
9
10
8
7
5
3
4
2
6
11
1
12
N
6
6
6
6
6
6
6
6
6
6
5
6
1
.01433
.01500
.01533
.01667
.02050
.02167
.02367
.02833
.03450
.03533
Subset
2
.02050
.02167
.02367
.02833
.03450
.03533
.04700
.04700
.06383
.999
.994
1.000
.406
1.000
1.000
1.000
.344
.000
.960
.999
.857
.945
.782
1.000
.473
.369
.299
.344
.037
.690
.003
.000
.000
.000
.028
.000
.000
.000
.000
.037
-.03423
-.03623
-.03307
-.04707
-.02923
-.02790
-.02690
-.04790
-.07640
-.04058
-.02057
-.01390
-.01590
-.01273
-.02673
-.00890
-.00757
-.00657
-.00723
-.05607
-.01208
.00793
.01460
.01260
.01577
.00177
.01960
.02093
.02193
.02127
.00093
.02090
.01890
.02207
.00807
.02590
.02723
.02823
.00723
-.02127
.01724
.03457
.04123
.03923
.04240
.02840
.04623
.04757
.04857
.04790
-.00093
.04574
.06307
.06973
.06773
.07090
.05690
.07473
.07607
.07707
.07640
.05607
19
Sig.
.310
.074
.637

The error term is Mean Square(Error) = .000.
b. The group sizes are unequal. The harmonic mean of the group
sizes is used. Type I error levels are not guaranteed.
c. Alpha = .05.
BIOFILM FORMATION ASSAY USING Staphylococcus aureus
ROSEMARY
Type III Sum of
Source
Squares
df
Mean Square
Corrected Model
.085a
23
.004
Intercept
.306
1
.306
Concentrations
.068
11
.006
Group
.000
1
.000
.017
11
.002
Error
.183
48
.004
Total
.574
72
Corrected Total
.268
71
F
.972
80.136
1.623
.034
.407
Sig.
.514
.000
.122
.854
.946

2. Multiple Comparison
Tukey HSD

1
2
3
4
5
6
7
8
9
10
11
12
2
1
3
4
5
6
7
8
9
Mean
Difference (IJ)
Std. Error
-.00350
.035655
-.03383
.035655
-.00250
.035655
-.00233
.035655
-.03283
.035655
-.00517
.035655
-.01050
.035655
-.00183
.035655
-.02933
.035655
-.01917
.035655
-.11483
.035655
.00350
.035655
-.03033
.035655
.00100
.035655
.00117
.035655
-.02933
.035655
-.00167
.035655
-.00700
.035655
.00167
.035655

Sig.
1.000
-.12593
.11893
.998
-.15626
.08860
1.000
-.12493
.11993
1.000
-.12476
.12010
.999
-.15526
.08960
1.000
-.12760
.11726
1.000
-.13293
.11193
1.000
-.12426
.12060
.999
-.15176
.09310
1.000
-.14160
.10326
.085
-.23726
.00760
1.000
-.11893
.12593
.999
-.15276
.09210
1.000
-.12143
.12343
1.000
-.12126
.12360
.999
-.15176
.09310
1.000
-.12410
.12076
1.000
-.12943
.11543
1.000
-.12076
.12410
20
10
11
12
1
2
4
5
6
7
8
9
10
11
12
1
2
3
5
6
7
8
9
10
11
12
1
2
3
4
6
7
8
9
10
11
12
1
2
3
4
5
7
8
9
10
11
12
1
2
3
4
-.02583
-.01567
-.11133
.03383
.03033
.03133
.03150
.00100
.02867
.02333
.03200
.00450
.01467
-.08100
.00250
-.00100
-.03133
.00017
-.03033
-.00267
-.00800
.00067
-.02683
-.01667
-.11233
.00233
-.00117
-.03150
-.00017
-.03050
-.00283
-.00817
.00050
-.02700
-.01683
-.11250
.03283
.02933
-.00100
.03033
.03050
.02767
.02233
.03100
.00350
.01367
-.08200
.00517
.00167
-.02867
.00267
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
1.000
1.000
.107
.998
.999
.999
.999
1.000
1.000
1.000
.999
1.000
1.000
.509
1.000
1.000
.999
1.000
.999
1.000
1.000
1.000
1.000
1.000
.100
1.000
1.000
.999
1.000
.999
1.000
1.000
1.000
1.000
1.000
.099
.999
.999
1.000
.999
.999
1.000
1.000
.999
1.000
1.000
.491
1.000
1.000
1.000
1.000
-.14826
-.13810
-.23376
-.08860
-.09210
-.09110
-.09093
-.12143
-.09376
-.09910
-.09043
-.11793
-.10776
-.20343
-.11993
-.12343
-.15376
-.12226
-.15276
-.12510
-.13043
-.12176
-.14926
-.13910
-.23476
-.12010
-.12360
-.15393
-.12260
-.15293
-.12526
-.13060
-.12193
-.14943
-.13926
-.23493
-.08960
-.09310
-.12343
-.09210
-.09193
-.09476
-.10010
-.09143
-.11893
-.10876
-.20443
-.11726
-.12076
-.15110
-.11976
.09660
.10676
.01110
.15626
.15276
.15376
.15393
.12343
.15110
.14576
.15443
.12693
.13710
.04143
.12493
.12143
.09110
.12260
.09210
.11976
.11443
.12310
.09560
.10576
.01010
.12476
.12126
.09093
.12226
.09193
.11960
.11426
.12293
.09543
.10560
.00993
.15526
.15176
.12143
.15276
.15293
.15010
.14476
.15343
.12593
.13610
.04043
.12760
.12410
.09376
.12510
21
10
11
5
6
8
9
10
11
12
1
2
3
4
5
6
7
9
10
11
12
1
2
3
4
5
6
7
8
10
11
12
1
2
3
4
5
6
7
8
9
11
12
1
2
3
4
5
6
7
8
9
10
12
.00283
-.02767
-.00533
.00333
-.02417
-.01400
-.10967
.01050
.00700
-.02333
.00800
.00817
-.02233
.00533
.00867
-.01883
-.00867
-.10433
.00183
-.00167
-.03200
-.00067
-.00050
-.03100
-.00333
-.00867
-.02750
-.01733
-.11300
.02933
.02583
-.00450
.02683
.02700
-.00350
.02417
.01883
.02750
.01017
-.08550
.01917
.01567
-.01467
.01667
.01683
-.01367
.01400
.00867
.01733
-.01017
-.09567
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
1.000
1.000
1.000
1.000
1.000
1.000
.119
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
.164
1.000
1.000
.999
1.000
1.000
.999
1.000
1.000
1.000
1.000
.096
.999
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
.427
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
.266
-.11960
-.15010
-.12776
-.11910
-.14660
-.13643
-.23210
-.11193
-.11543
-.14576
-.11443
-.11426
-.14476
-.11710
-.11376
-.14126
-.13110
-.22676
-.12060
-.12410
-.15443
-.12310
-.12293
-.15343
-.12576
-.13110
-.14993
-.13976
-.23543
-.09310
-.09660
-.12693
-.09560
-.09543
-.12593
-.09826
-.10360
-.09493
-.11226
-.20793
-.10326
-.10676
-.13710
-.10576
-.10560
-.13610
-.10843
-.11376
-.10510
-.13260
-.21810
.12526
.09476
.11710
.12576
.09826
.10843
.01276
.13293
.12943
.09910
.13043
.13060
.10010
.12776
.13110
.10360
.11376
.01810
.12426
.12076
.09043
.12176
.12193
.09143
.11910
.11376
.09493
.10510
.00943
.15176
.14826
.11793
.14926
.14943
.11893
.14660
.14126
.14993
.13260
.03693
.14160
.13810
.10776
.13910
.13926
.10876
.13643
.13110
.13976
.11226
.02676
22
12
1
.11483
2
.11133
3
.08100
4
.11233
5
.11250
6
.08200
7
.10967
8
.10433
9
.11300
10
.08550
11
.09567
3.
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.035655
.085
.107
.509
.100
.099
.491
.119
.164
.096
.427
.266
-.00760
-.01110
-.04143
-.01010
-.00993
-.04043
-.01276
-.01810
-.00943
-.03693
-.02676
Homogeneous subsets
Concentrations
1
9
5
4
2
7
8
11
10
6
3
12
Sig.
N
6
6
6
6
6
6
6
6
6
6
6
6
Subset
1
.04383
.04567
.04617
.04633
.04733
.04900
.05433
.06300
.07317
.07667
.07767
.15867
.085

b. Alpha = .05.
SAGE
Type III Sum of
Source
Squares
Corrected Model
.094a
Intercept
.279
Concentrations
.069
Group
5.000E-7
.024
Error
.175
Total
.547
Corrected Total
.269
df
23
1
11
1
11
48
72
71
Mean Square
.004
.279
.006
5.000E-7
.002
.004
F
1.116
76.382
1.733
.000
.601
Sig.
.364
.000
.094
.991
.818
.23726
.23376
.20343
.23476
.23493
.20443
.23210
.22676
.23543
.20793
.21810
23
5.
Multiple Comparison
Multiple Comparisons

Tukey HSD

1
2
3
4
5
6
7
8
9
10
11
12
2
1
3
4
5
6
7
8
9
10
11
12
3
1
2
4
5
6
7
8
9
10
11
12
4
1
2
3
5
6
7
8
9
10
11
Mean
Difference (IJ)
Std. Error
-.00350
.034863
-.01883
.034863
-.00250
.034863
-.00133
.034863
-.01917
.034863
-.00517
.034863
-.01050
.034863
-.00183
.034863
-.02933
.034863
-.01083
.034863
-.11733
.034863
.00350
.034863
-.01533
.034863
.00100
.034863
.00217
.034863
-.01567
.034863
-.00167
.034863
-.00700
.034863
.00167
.034863
-.02583
.034863
-.00733
.034863
-.11383
.034863
.01883
.034863
.01533
.034863
.01633
.034863
.01750
.034863
-.00033
.034863
.01367
.034863
.00833
.034863
.01700
.034863
-.01050
.034863
.00800
.034863
-.09850
.034863
.00250
.034863
-.00100
.034863
-.01633
.034863
.00117
.034863
-.01667
.034863
-.00267
.034863
-.00800
.034863
.00067
.034863
-.02683
.034863
-.00833
.034863

Sig.
1.000
-.12321
.11621
1.000
-.13854
.10088
1.000
-.12221
.11721
1.000
-.12104
.11838
1.000
-.13888
.10054
1.000
-.12488
.11454
1.000
-.13021
.10921
1.000
-.12154
.11788
.999
-.14904
.09038
1.000
-.13054
.10888
.059
-.23704
.00238
1.000
-.11621
.12321
1.000
-.13504
.10438
1.000
-.11871
.12071
1.000
-.11754
.12188
1.000
-.13538
.10404
1.000
-.12138
.11804
1.000
-.12671
.11271
1.000
-.11804
.12138
1.000
-.14554
.09388
1.000
-.12704
.11238
.076
-.23354
.00588
1.000
-.10088
.13854
1.000
-.10438
.13504
1.000
-.10338
.13604
1.000
-.10221
.13721
1.000
-.12004
.11938
1.000
-.10604
.13338
1.000
-.11138
.12804
1.000
-.10271
.13671
1.000
-.13021
.10921
1.000
-.11171
.12771
.202
-.21821
.02121
1.000
-.11721
.12221
1.000
-.12071
.11871
1.000
-.13604
.10338
1.000
-.11854
.12088
1.000
-.13638
.10304
1.000
-.12238
.11704
1.000
-.12771
.11171
1.000
-.11904
.12038
1.000
-.14654
.09288
1.000
-.12804
.11138
24
12
1
2
3
4
6
7
8
9
10
11
12
1
2
3
4
5
7
8
9
10
11
12
1
2
3
4
5
6
8
9
10
11
12
1
2
3
4
5
6
7
9
10
11
12
1
2
3
4
5
6
-.11483
.00133
-.00217
-.01750
-.00117
-.01783
-.00383
-.00917
-.00050
-.02800
-.00950
-.11600
.01917
.01567
.00033
.01667
.01783
.01400
.00867
.01733
-.01017
.00833
-.09817
.00517
.00167
-.01367
.00267
.00383
-.01400
-.00533
.00333
-.02417
-.00567
-.11217
.01050
.00700
-.00833
.00800
.00917
-.00867
.00533
.00867
-.01883
-.00033
-.10683
.00183
-.00167
-.01700
-.00067
.00050
-.01733
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.071
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
.065
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
.206
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
.085
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
.122
1.000
1.000
1.000
1.000
1.000
1.000
-.23454
-.11838
-.12188
-.13721
-.12088
-.13754
-.12354
-.12888
-.12021
-.14771
-.12921
-.23571
-.10054
-.10404
-.11938
-.10304
-.10188
-.10571
-.11104
-.10238
-.12988
-.11138
-.21788
-.11454
-.11804
-.13338
-.11704
-.11588
-.13371
-.12504
-.11638
-.14388
-.12538
-.23188
-.10921
-.11271
-.12804
-.11171
-.11054
-.12838
-.11438
-.11104
-.13854
-.12004
-.22654
-.11788
-.12138
-.13671
-.12038
-.11921
-.13704
.00488
.12104
.11754
.10221
.11854
.10188
.11588
.11054
.11921
.09171
.11021
.00371
.13888
.13538
.12004
.13638
.13754
.13371
.12838
.13704
.10954
.12804
.02154
.12488
.12138
.10604
.12238
.12354
.10571
.11438
.12304
.09554
.11404
.00754
.13021
.12671
.11138
.12771
.12888
.11104
.12504
.12838
.10088
.11938
.01288
.12154
.11804
.10271
.11904
.12021
.10238
25
10
11
12
7
8
10
11
12
1
2
3
4
5
6
7
8
9
11
12
1
2
3
4
5
6
7
8
9
10
12
1
2
3
4
5
6
7
8
9
10
11
-.00333
-.00867
-.02750
-.00900
-.11550
.02933
.02583
.01050
.02683
.02800
.01017
.02417
.01883
.02750
.01850
-.08800
.01083
.00733
-.00800
.00833
.00950
-.00833
.00567
.00033
.00900
-.01850
-.10650
.11733
.11383
.09850
.11483
.11600
.09817
.11217
.10683
.11550
.08800
.10650
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863
.034863

Absorbances
Tukey HSDa,b,c
Concentrations
1
5
9
Subset
1
N
6
6
6
.04383
.04517
.04567
1.000
1.000
1.000
1.000
.068
.999
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
.350
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
.124
.059
.076
.202
.071
.065
.206
.085
.122
.068
.350
.124
-.12304
-.12838
-.14721
-.12871
-.23521
-.09038
-.09388
-.10921
-.09288
-.09171
-.10954
-.09554
-.10088
-.09221
-.10121
-.20771
-.10888
-.11238
-.12771
-.11138
-.11021
-.12804
-.11404
-.11938
-.11071
-.13821
-.22621
-.00238
-.00588
-.02121
-.00488
-.00371
-.02154
-.00754
-.01288
-.00421
-.03171
-.01321
.11638
.11104
.09221
.11071
.00421
.14904
.14554
.13021
.14654
.14771
.12988
.14388
.13854
.14721
.13821
.03171
.13054
.12704
.11171
.12804
.12921
.11138
.12538
.12004
.12871
.10121
.01321
.23704
.23354
.21821
.23454
.23571
.21788
.23188
.22654
.23521
.20771
.22621
26
4
2
7
8
11
3
6
10
12
Sig.
6
6
6
6
6
6
6
6
6
.04633
.04733
.04900
.05433
.05467
.06267
.06300
.07317
.16117
.059

The error term is Mean Square(Error) = .000.
b. The group sizes are unequal. The harmonic mean of the group
sizes is used. Type I error levels are not guaranteed.
c. Alpha = .05.
ANTIBIOTIC RESISTANCE ASSAY
CEFOXITIN
ROSEMARY
MRSA1 MRSA2 MRSA3
Rosemar
19
18
19
y
21
18
18
20
18
19
Pos
12
11
18
12
11
18
12
11
18
Neg
21
6
21
22
6
22
23
6
23
ANOVA: Two-Factor with Replication
SUMMARY MRSA1 MRSA2
Rosemary
Count
3
3
Sum
60
54
Average
20
18
Variance
1
0
Pos
Count
3
3
Sum
36
33
Average
12
11
Variance
0
0
Neg
Count
3
3
Sum
66
18
Average
22
6
MRSA4
22
22
22
20
20
20
6
6
6
MRSA3
SA1
SA2
29
30
30
28
28
28
32
30
30
MRSA4
SA1
SA3
SA4
29
31
30
29
29
28
6
6
6
24
25
25
20
25
25
32
30
30
40
41
40
28
28
28
6
6
6
SA2
SA3
SA4
Total
3
56
18.66667
0.333333
3
66
22
0
3
89
29.66667
0.333333
3
90
30
1
3
74
24.66667
0.333333
3
121
40.33333
0.333333
24
610
25.41667
52.94928
3
54
18
0
3
60
20
0
3
84
28
0
3
86
28.66667
0.333333
3
70
23.33333
8.333333
3
84
28
0
24
507
21.125
46.80978
3
66
22
3
18
6
3
92
30.66667
3
18
6
3
92
30.66667
3
18
6
24
388
16.16667
27
Variance
Total
Count
Sum
Average
Variance
ANOVA
Source of
Variation
Sample
Columns
Interaction
Within
Total
1.333333
1.333333
9
162
18
21.5
9
105
11.66667
27.25
9
176
19.55556
3.777778
9
144
16
57
9
265
29.44444
1.777778
9
194
21.55556
136.7778
9
236
26.22222
13.94444
9
223
24.77778
226.9444
SS
df
1028.528
2126.542
2849.917
33.33333
2
7
14
48
6038.319
71
MS
P-value
F crit
514.2639
303.7917
203.5655
0.694444
740.54
437.46
293.1343
8.38E-37
3.25E-41
1.8E-41
3.190727
2.207436
1.903653
118.058
SAGE
Sage
Pos
Neg
MRSA MRSA MRSA MRSA

1
2
3
4
SA1
SA2
SA3
SA4
21
20
19
17
29
31
30
28
20
19
19
19
29
31
30
30
20
19
18
18
29
31
30
30
12
11
18
20
28
29
20
28
12
11
18
20
28
29
25
28
12
11
18
20
28
28
25
28
21
6
21
6
32
6
32
6
22
6
22
6
30
6
30
6
23
6
23
6
30
6
30
6

SUMMARY
Sage
Count
Sum
Average
Variance
Pos
Count
Sum
Average
Variance
Neg
Count
Sum
MRSA1
MRSA2
MRSA3
MRSA4
SA1
SA2
SA3
SA4
Total
3
61
20.33333
0.333333
3
58
19.33333
0.333333
3
56
18.66667
0.333333
3
54
18
1
3
87
29
0
3
93
31
0
3
90
30
0
3
88
29.33333
1.333333
24
587
24.45833
31.12862
3
36
12
0
3
33
11
0
3
54
18
0
3
60
20
0
3
84
28
0
3
86
28.66667
0.333333
3
70
23.33333
8.333333
3
84
28
0
24
507
21.125
46.80978
3
66
3
18
3
66
3
18
3
92
3
18
3
92
3
18
24
388
28
Average
Variance
Total
Count
Sum
Average
Variance
22
1
6
0
22
1
6
0
30.66667
1.333333
6
0
30.66667
1.333333
6
0
9
163
18.11111
21.86111
9
109
12.11111
34.11111
9
176
19.55556
3.777778
9
132
14.66667
43.25
9
263
29.22222
1.694444
9
197
21.88889
143.1111
9
252
28
14.75
9
190
21.11111
129.1111
16.16667
118.058
ANOVA
Source of
Variation
Sample
SS
df
MS
P-value
835.5833
417.7917
601.62
Columns
2210.167
315.7381
454.6629
Interaction
2264.417
14
161.744
232.9114
Within
Total
33.33333
5343.5
48
71
0.694444
F crit
1.03E34
1.31E41
4.12E39
3.190727
2.207436
1.903653
OXACILLIN
ROSEMARY
Rosema
ry
Pos
Neg
TSB1
TSB2
TSB3
TSB4
0.64
0.646
0.702
0.574
0.6
0.588
0.784
0.757
0.775
0.62
0.627
0.619
0.528
0.499
0.525
0.784
0.757
0.775
0.772
0.696
0.704
0.72
0.727
0.777
0.784
0.757
0.775
0.542
0.445
0.513
0.681
0.744
0.775
0.784
0.757
0.775
ANOVA: TWO-FACTOR WITH REPLICATION

SUMMAR
TSB1
TSB2
TSB3
Y
Rosemary
Count
3
3
3
Sum
1.988
1.866
2.172
Average
0.66266
0.622
0.724
7
Variance
0.00116
0.00001
0.00174
9
9
4
Pos
Count
3
3
3
TSB+Ox TSB+Ox TSB+Ox TSB+Ox

a1
a2
a3
a4
0
0.101
0.028
0.004
0.015
0.116
0.025
0
0.033
0.266
0.047
0.007
0
0.617
0.06
0.027
0.174
0.635
0.042
0.077
0.377
0.589
0.063
0.083
0.685
0.685
0.685
0.685
0.625
0.625
0.625
0.625
0.678
0.678
0.678
0.678
TSB4
TSB+Oxa
1
TSB+Oxa
2
TSB+Oxa
3
TSB+Oxa
4
Total
3
1.5
0.5
3
0.048
0.016
3
0.483
0.161
3
0.1
0.033333
3
0.011
0.003667
0.00247
9
0.000273
0.008325
0.000142
1.23E-05
24
8.168
0.34033
3
0.09265
24
29
Sum
Average
1.762
0.58733
3
0.00016
9
1.552
0.51733
3
0.00025
4
2.224
0.74133
3
0.00096
6
2.2
0.73333
3
0.00229
4
0.551
0.183667
1.841
0.613667
0.165
0.055
0.187
0.062333
10.482
0.43675
0.035602
0.000537
0.000129
0.000945
0.08066
6
Count
Sum
Average
3
2.316
0.772
3
2.316
0.772
3
2.316
0.772
3
2.316
0.772
3
1.988
0.662667
3
1.988
0.662667
3
1.988
0.662667
3
1.988
0.662667
Variance
0.00018
9
0.00018
9
0.00018
9
0.00018
9
0.001076
0.001076
0.001076
0.001076
24
17.216
0.71733
3
0.00355
8
Count
Sum
Average
9
6.066
0.674
9
5.734
0.637111
9
4.312
0.479111
9
2.253
0.250333
9
2.186
0.242889
0.00684
8
0.01240
4
9
6.016
0.66844
4
0.01748
1
9
2.587
0.287444
Variance
9
6.712
0.74577
8
0.00116
8
0.093704
0.059857
0.096061
0.100274
P-value
7.94E-30
4.88E-31
1.06E-19
F crit
3.190727
2.207436
1.903653
Variance
Neg
Total
ANOVA
Source of Variation
Sample
Columns
Interaction
Within
Total
SS
1.841217
2.806954
1.140912
0.120247
df
MS
0.920609
0.400993
0.081494
0.002505
2
7
14
48
5.909331
F
367.4861
160.0675
32.53043
71
SAGE
TSB1
Sage
Pos
Neg
0.637
0.073
0.704
0.574
0.6
0.588
0.784
0.757
0.775
TSB2
0.602
0.607
0.552
0.528
0.499
0.525
0.784
0.757
0.775
TSB3
TSB4
0.736
0.735
0.765
0.72
0.727
0.777
0.784
0.757
0.775

SUMMAR TSB1
TSB2
TSB3
Y
Sage
Count
3
3
3
Sum
1.414
1.761
2.236
0.719
0.724
0.992
0.681
0.744
0.775
0.784
0.757
0.775
TSB+Oxa
1
0.012
0.058
0.099
0
0.174
0.377
0.685
0.625
0.678
TSB4
3
2.435
TSB+Oxa
2
0.023
0.002
0.26
0.617
0.635
0.589
0.685
0.625
0.678
TSB+Oxa
3
0.132
0.074
0.017
0.06
0.042
0.063
0.685
0.625
0.678
TSB+Oxa
4
0.001
0.031
0.046
0.027
0.077
0.083
0.685
0.625
0.678
TSB+Oxa
1
TSB+Oxa
2
TSB+Oxa
3
TSB+Oxa
4
3
0.169
3
0.285
3
0.223
3
0.078
Total
24
8.601
30
Average
0.47133
3
0.12012
4
0.00092
5
3
1.762
0.58733
3
0.00016
9
Count
Sum
Average
Variance
Variance
Pos
Count
Sum
Average
Variance
0.587
0.74533
3
0.00029
0.81166
7
0.02439
6
0.056333
0.095
0.074333
0.026
0.001894
0.020529
0.003306
0.000525
0.35837
5
0.11565
3
1.552
0.51733
3
0.00025
4
3
2.224
0.74133
3
0.00096
6
3
2.2
0.73333
3
0.00229
4
3
0.551
0.183667
3
1.841
0.613667
3
0.165
0.055
3
0.187
0.062333
24
10.482
0.43675
0.035602
0.000537
0.000129
0.000945
0.08066
6
3
2.316
0.772
3
2.316
0.772
3
2.316
0.772
3
2.316
0.772
3
1.988
0.662667
3
1.988
0.662667
3
1.988
0.662667
3
1.988
0.662667
0.00018
9
0.00018
9
0.00018
9
0.00018
9
0.001076
0.001076
0.001076
0.001076
24
17.216
0.71733
3
0.00355
8
9
5.492
0.61022
2
0.04736
5
9
5.629
0.62544
4
0.01333
4
9
6.776
0.75288
9
0.00057
9
6.951
0.77233
3
0.00787
1
9
2.708
0.300889
9
4.114
0.457111
9
2.376
0.264
9
2.253
0.250333
0.086305
0.079743
0.090599
0.09652
MS
Neg
Total
Count
Sum
Average
Variance
ANOVA
Source of
Variation
Sample
Columns
Interaction
Within
Total
SS
1.70976
6
2.92841
7
1.23278
8
0.43590
1
6.30687
1
df
2
7
14
48
71
0.85488
3
0.41834
5
0.08805
6
0.00908
1
94.137
46.0668
6
9.69647
8
P-value
2.44E17
3.64E19
1.07E09
F crit
3.19072
7
2.20743
6
1.90365
3
31
APPENDIX B
BIOFILM FORMATION ASSAY
After incubation at 37oC
After incubation at 37oC
Decanting of trypticase soy

broth with running water
32
Three times washing with 200

l of distilled water
Staining with 200 l of crystal

violet and stand for 45
minutes
Decanting of stain with

running water after standing
for 45 minutes
33
Blot drying after staining
After blot drying
Dissolving stain with 200 l of

95:5 ethanol acetic acid
34
Mixing the sample and the

95:5 ethanol acetic acid (5
times)
Transferring the solution to

tubes
Reading
spectrophotometrically at 570
nm and recording of result of
reading
35
Reading
spectrophotometrically at 545
nm, 580 nm, differential
reading (545-580 nm) and
recording of result of reading
ANTIBIOTIC RESISTANCE ASSSAY
Supplementing the
microorganisms with the 100
l of extracts
Performing aseptic technique

for inoculating the
microorganisms into Mueller
Hinton Agar
36
Getting 2-3 loopfull of

inoculum to be inoculated to
Mueller Hinton Agar
Inoculation into Mueller Hinton

Agar

after inoculation
37
Getting inoculum to be
swabbed to Mueller Hinton
Agar
Swabbing to Mueller Hinton

Agar

for disc diffusion method
38
Proper technique for getting

discs
Proper placing of discs to the

culture medium with
microorganisms

after placing the discs
39
Appendix C: GAANT Chart (Timetable)
Activities
Research
topic
Gathering of
data for
proposal
Introduction
Revision of
Introduction
Methodolog
y
Revision of
methodology
Proposal
Conducting
Experiment
Gathering of
Results
Treatment of
Data
Finalizing of
Research
Defense
Aug
Sep
Oct
Nov
Dec
Jan
Feb
Mar
Apr
May
40
Appendix D: Budgetary Requirement
Item
Particulars
Price
mecA-positive methicillinresistant Staphylococcus

aureus (MRSA) ATCC 43300
1 lot lyophilized bacterial

cells
PhP 1,900.00
mecA-negative
Staphylococcus aureus ATCC
25923

cells
PhP 1,900.00
Pseudomonas aeruginosa
PA01

cells
PhP 1,900.00
C. violaceum CV026

cells
PhP 1,900.00
C. violaceum ATCC 31532

cells
PhP 1,900.00
Mueller Hinton Agar
1 500g-pack
PhP 980.00
Tryptic Soy Broth
1 500g-pack
PhP 980.00
Cefoxitin discs
2 50pcs-package
PhP 600.00
Microtiter plates
10 pieces
PhP 500.00
Miscellaneous reagents and

supplies
PhP 1,500.00
TOTAL
PhP 14,060.00

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ANTI-QUORUM SENSING ACTIVITY OF ROSEMARY (Rosmarinus officinalis) AND

SAGE (Salvia officinalis) LEAF EXTRACTS AGAINST METHICILLIN-RESISTANT

In Partial Fulfillment of the Requirements

S1: ASISTIN, RALPH JUSTINE A.

S5: REYES, EDGAR JR. S.

(signature over printed name)

(signature over printed name)

S2: CHIOK, JERELYNE T.

S6: TERRADO, MELVIN JETHRO E.

(signature over printed name)

(signature over printed name)

S3: FELICIANO, REYNOLD G.

S7: VENTEREZ, GEENA LINDSAY F.

(signature over printed name)

S4: NAVARRO, ANGELICA MAE B.

(signature over printed name)

(signature over printed

(signature over printed name)

Date: June 9, 2016

JENNIFER T. BUYA RMT

In partial fulfillment of the requirements for the degree,

ANTI- QUORUM SENSING ACTIVITY OF ROSEMARY (Rosmarinus officinalis) AND

prepared and submitted by:

S5: REYES, EDGAR JR. S.

(signature over printed name)

(signature over printed name)

S2: CHIOK, JERELYNE T.

S6: TERRADO, MELVIN JETHRO E.

(signature over printed name)

(signature over printed name)

S3: FELICIANO, REYNOLD G.

S7: VENTEREZ, GEENA LINDSAY F.

(signature over printed name)

S4: NAVARRO, ANGELICA MAE B.

(signature over printed name)

(signature over printed

(signature over printed name)

is hereby endorsed for evaluation and approval.

JENNIFER T. BUYA RMT

prepared and submitted by

S5: REYES, EDGAR JR. S.

(signature over printed name)

(signature over printed name)

S2: CHIOK, JERELYNE T.

S6: TERRADO, MELVIN JETHRO E.

(signature over printed name)

(signature over printed name)

S3: FELICIANO, REYNOLD G.

S7: VENTEREZ, GEENA LINDSAY F.

(signature over printed name)

S4: NAVARRO, ANGELICA MAE B.

(signature over printed name)

(signature over printed

(signature over printed name)

JENNIFER T. BUYA RMT

SANDRA LYN D. DELA PEA

Allan Jay C. Espiritu, RMT, M.Sc.

a. R Squared = .925 (Adjusted R Squared = .889)

(I) Concentrations (J) Concentrations

95% Confidence Interval

Means for groups in homogeneous subsets are displayed.

a. R Squared = .691 (Adjusted R Squared = .540)

Dependent Variable: Absorbances