UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 1

CHAPTER 1

THE PROBLEM RATIONALE

1.1 Rationale

Despite advances in antibiotic therapy, antibiotic resistance continue to

contribute to high mortality and morbidity among hospitalized patients (Valle Jr,

et al. 2015). Antibiotic resistance and multidrug resistant (AMR) are some

problems that continue to challenge a large part of the healthcare sector and

remains an unresolved problem of the world.

Some medical practitioners resort to the use of second- or third choice of

drugs to solve the problem of AMR that may subject the patients to a higher risk

due to the possibility of having more harmful side effects. Other ways to this

include controlling the use of antibiotics, understanding the mechanisms of

resistance and developing new antibiotics by identifying new sources of natural

products with antimicrobial activities, and promoting chemical diversity that leads

to discovering new drugs (Valle Jr, et al. 2015).

Methicillin-resistant S. aureus or MRSA are strains of S. aureus that

through the process of natural selection developed resistance to all available

penicillins and other β-lactam antimicrobial drugs (Daum, 2010). MRSA has

become a major problem in the hospital and in the community setting (San Juan

et al. 2012). MRSA through contaminated objects, casual contact can be spread
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 2

from one person to another. In the Philippines, greater than 30% of community-

acquired S. aureus (CASA) and 38% of hospital-acquired S. aureus (HASA)

infections are caused by MRSA (Song et al. 2011).

According to Pantosti & Venditti (2009), community-acquired Methicillin-

resistant S. aureus (CA)-MRSA have epidemiological, clinical and

microbiological features that differentiate them from hospital-acquired

Methicillin-resistant S. aureus (HA)-MRSA, although each characteristic is not

exclusive of each group. Typically, CA-MRSA strains cause skin and mostly

recurrent soft tissue infections, including furuncles, abscesses, impetigo and

cellulitis.

Several published works concerning the significance of traditional

medicinal plants as alternatives to synthetic antimicrobial and antifungal come

from countries that still practice the use of herbal medicine for the treatment of

numerous diseases for practical reasons. These studies are vital for local medical

scientists who are seeking to explore the antimicrobial activities of the Philippine

medicinal plants, most particularly against methicillin-resistant Staphylococcus

aureus (Valle Jr, et al. 2015). Medicinal plants have been known to be an

abundant source of remedies for different diseases and wide arrays of therapeutic

potential of antimicrobial plants have been found to be effective in treating

infectious diseases.
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 3

According to Valle J et al. (2015), the Philippines is one of the Asian

countries with a diverse flora, and numerous species that possesses curative

properties. However, these claims lack scientific value and further studies, one of

these is Piper betle (P. betle). P. betle is a multifunctional medicinal plant that is

cultivated throughout the Philippines. The leaf extracts of P. betle showed

antibacterial activity against Gram-positive methicillin-resistant S. aureus and

vancomycin-resistant Enterococcus. It showed a 16-33 mm inhibition diameter in

the disk diffusion, minimum inhibitory concentration of 19-156 µg/mL, and

minimum bactericidal concentration of 312-625 µg/mL. Furthermore, it showed

remarkable antibacterial activity for all the Gram-negative multidrug-resistant

bacteria including: extended spectrum β-lactamase-producing, carbapenem-

resistant Enterobacteriaceae and metallo-β-lactamase. Producing an inhibition

diameter of 17–21 mm, a minimum inhibitory concentration of 312-625 µg/mL

and a minimum inhibitory concentration of 312-625 µg/mL in disk diffusion.

Some studies also show that the plant extract of P. betle is the most

effective antidermatophytic against human pathogenic dermatophytes which

includes Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum

gypseum and Candida albicans (Bhadauria S., & Kumar P. 2011). However, given

these studies, there are still no formulated dosage forms available.

This study aimed to evaluate and enhance the functionality of the

antimicrobial activity of ethanolic leaf extract of Piper betle against methicillin-

 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 4

resistant Staphylococcus aureus and to formulate a topical cream containing the

extract that can be potentially added in the market.

1.2 Research Impediments

The study evaluated the antimicrobial properties of the cream containing

ethanolic leaf extract of P. betle via in vitro testing. Formulation studies were

done to determine the physico-chemical (organoleptic characteristics, pH value

determination, viscosity, spreadability, and phase separation study), stability and

compatibility of the ingredients and P. betle ethanolic leaf extract. Furthermore,

this study sought the effects of one plant part only, specifically the leaves of P.

betle.

The study is limited on the potentiality of P. betle ethanolic extract using

cream only as its delivery system. Determination of the effect on other strains of

S. aureus and other organisms were not included in this study, thus the product

may only be effective on one type of infection. Assay of the ethanolic leaf extract

and potential antimicrobial activity of the other parts of the plant were not

assessed within this study.

 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 5

CHAPTER 2

THE RESEARCH QUESTIONS

This chapter presents a review of pertinent literature on Piper betle,

antimicrobial resistance, methicillin-resistant Staphylococcus aureus, topical

formulation and stability studies. The research questions to be answered in the

study are also outlined.

2.1. Piper betle

2.1.1. Taxonomical classification

The following presents the taxonomic classification of Piper betle from

Integrated Taxonomic Information System (ITIS):

Plantae - Kingdom
Viridiplantae - Subkingdom
Streptophyta - Infrakingdom
Embryophyta - Superdivision
Tracheophyta - Division
Spermatophytina - Subdivision
Magnoliopsida - Class
Magnolianae - Superorder
Piperales - Order
Piperaceae - Family
Piper L. - Genus
Piper betle L. - Species
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 6

2.1.1.2 Botanical description

The Betel pepper or ikmo is a dioecious, perennial, woody plant that is 5-

20 m long. Its orthotropic branches vegetative, bearing roots; plagiotropic

branches generative, bearing no roots. Leaves are shiny bright green, alternate and

coriaceous; petiole 1-2.5 cm long, blade ovate to ovate oblong; cordate base,

rounded or oblique; margin entire, apex acuminate. Inflorescence a cylindrical,

pendulous spike; male spike up to 12 cm long, with flowers and 2 stamens;

female spike up to 5 cm x 5 mm, crowded with female flowers with 3-5 stigmas.

Its fruit a fleshy drupe, and its seed suborbicular, 3-5 mm in diameter. (Banka &

Teo, 2016)

2.1.2 Phytochemical Composition

Piper betle is one of the highly investigated plants and their

phytochemical studies show that the plant contains a wide variety of biologically

active compounds (Table 1).

Table 1. Phytochemical constituents of Piper betle

Plant Part Constituents Uses Results & Reference

 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 7

Leaves Eugenol Antihypercholesterole- The

mic antihypercholesterol
emic properties of an
ethanolic extract of
Piper betle and its
active constituent,
eugenol, were
evaluated in
experimental
hypercholesterolemi
a in Wistar rats.
Results showed that
eugenol possesses
antihyper-
cholesterolemic
properties
(Venkadeswaran et
al., 2014).

Antibacterial The methanolic

extracts of P. betle
leaves were
identified for its
antibacterial active
compounds with
reference to the
major compounds:
hydroxychavicol and
eugenol against 9
species of Gram
positive and Gram-
negative bacteria.
Results verified that
two pure compounds
from P. betle were
antibacterial active
compounds with
promising
antibacterial activity
(Syahidah et al.,
2017).
UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 8

The study isolated

and identified the
antimicrobial
compounds of Piper
betle L. leaf ethanol
extracts by thin layer
chromatography-
(TLC-)
bioautography and
gas chromatography-
mass spectrometry
(GC-MS). GC-MS
identified six
compounds
including eugenol
which showed
antibacterial
activities against
various plant and
human pathogens,
including MRSA. Its
mechanism of action
is reported to be the
deformation of
macromolecules in
the cytoplasmic
membrane as
verified by FT-IR
spectroscopy (Valle
Jr. et al., 2016).
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 9

Hydroxychavicol Antimutagenic Hydroxychavicol

(HC) was separated from
betel leaf extract
using C18 phase
bonded Hiflosil
silica gel. It
exhibited dose-
dependent
suppression of
dimethylbenzanthrac
ene- induced
mutagenesis in S.
typhimurium strain
TA98 with metabolic
activation (Amonkar
et al., 1986).

Antifungal Hydroxychavicol
was isolated from
the chloroform
extract of the
aqueous leaf extract
of Piper betle (Ali et
al., 2010).

Allylpyrocatechol Anti-halitosis Methanol extract of

(APC) P. betle leaves (500
µg/g) inhibited
obligate oral
anaerobes using
broth (86%) and
plate dilution (71%)
assays as compared
to chlorhexidine.
APC was found to
be the active
constituent (Ramji et
al., 2002).

Aristololactam A- Antifertility Aristololactam A-II

Roots II was isolated using
silica gel column
chromatography of
the alcoholic extract
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 10

of Piper Betle roots

(Ghosh &
Bhattacharya, 2005).

Stem Tannins Anthelmintic Phytochemical

analysis showed the
presence of tannins
and flavonoids in the
ethanolic extract of
the stems of Piper
Betle. Tannins were
shown to produce
anthelmintic
activities (Adate et
al., 2016).

2.1.3 Pharmacological studies

The following are the biological uses of the plant parts of Piper Betle in

some previous studies (Table 2).

Table 2. Pharmacologic Studies of Piper betle

Plant Part Biological Use Results & Reference
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 11

Leaves Antioxidant Leaf extract showed significant

Cytotoxic DPPH free radical scavenging
effect compared to standard
antioxidant ascorbic acid. IC50
value of ascorbic acid and leaf
extract was found 1.81μg/ml and
151.36μg/ml respectively. In
brine shrimp lethality bioassay
LC50 value of Piper betle
ethanol extract was found
274.63862μg/ml with 95%
confidence limit where the lower
and upper limits were 198.39
μg/ml and 387.18μg/ml
respectively, which indicates that
the Piper betle leaf extract has
promising cytotoxic effect (Uddin
et al., 2015).

Antifungal Ali et al. (2010) reported on the

in vitro antifungal activity of
hydroxychavicol (HC) isolated
from P. betle involving a total of
124 strains of selected fungi. HC
exhibited inhibitory effect on
fungal species with minimum
inhibitory concentrations ranging
from 15.62 to 500 μg/ml for
yeasts, 125 to 500 μg/ml for
Aspergillus species, and 7.81 to
62.5 μg/ml for dermatophytes
where as the minimum fungicidal
concentrations were found to be
similar or two fold greater than
the MICs.
UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 12

Antimicrobial Four different extracts (water,

Antioxidative methanol, ethyl acetate and
Antihemolytic petroleum ether) of Piper betle
leaves were tested against
pathogenic bacteria namely
Streptococcus pyogenes,
Staphylococcus aureus, Proteus
vulgaris and Escherichia coli.
Five concentrations of the
extracts were taken, (5, 10, 25,
50 and 100 mg mL‐1) and tested
against the four bacteria.

Anti‐oxidative studies were done

by TBARS and DPPH method.
Anti‐haemolytic activity was
determined using erythrocytes
model and the extent of lipid
peroxidation of the same was also
determined. The antioxidative &
antihemolytic activities were
attributed to the high
concentration & combined
activity of flavonoids &
polyphenols (Chakraborty &
Shah 2011).

Antimicrobial Caburian & Osi (2010) reported

that the essential oil obtained
from P. betle leaves were found
to have antimicrobial activity
against Staphylococcus aureus,
Streptococcus pyogenes, Candida
albicans, and Trichophyton
mentagrophytes with minimum
inhibitory concentration of 125
μg/mL, 15.60 μg/mL, 250 μg/mL
and 1.95 μg/mL respectively.
UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 13

Ethanol extract of P. betle leaves

was evaluated against
Pseudomonas aeruginosa,
Klebsiella pneumonia, Proteus
vulgaris and Staphylococcus
aureus using disc diffusion assay
with ceftriaxone (1 mg/mL) as
standard. MIC of the Proteus
vulgaris was found to be least (25
μg) while for S. aureus, P.
aeruginosa and Klebsiella
pneumonia were approximately
40, 35 and 25 μg (Datta et al.,
2011).

Antihistaminic Hajare, Davhekar, Shewale and

Patil (2010) studied that the
ethanol extract and essential oil
(100,200 mg/kg) of P. betle
leaves leaves administered orally
to Dunkin-Hartley guinea pigs
protected them against histamine-
induced bronchospasm producing
progressive dyspnea leading to
convulsions (64.5-77.48%) as
compared to chlorpheniramine
maleate (91.10%).

Antileishmanial Treatment of promastigotes with

P. betle ethanol extract (0-20
µg/mL) reduced the viability of
promastigotes (IC50 9.8 µg/mL)
in a dose-dependent manner by
inhibition of parasitic growth
using MTS-PMS assay.

The ethanol extract also reduce

the infection of amastigotes
(IC50 = 5.45 µg/mL) in a dose
dependent manner by inhibition
of parasitic load in peritoneal
macrophages from BALB/c mice
(Sarkar et al., 2008).
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 14

Stems Wound healing The animals treated with

ointment formulation containing
10% Piper betle
stem showed significant results
when compared with povidone
iodine and control group. From
Nilugal et al. (2014)
investigations concluded that
Piper betle has the potential
ability in wound healing.

Anthelmintic Adate et al. (2016) stated that the

ethanolic extract (50 mg/mL) of
stems of Piper betle showed
potent anthelminic activity as
compared to the standard drug
Albendazole (40 mg/mL) and the
aqueous extract (50 mg/mL). The
ethanolic extract did not only
demonstrated anthelmintic
property but they also caused the
death of the worms.

2.2 Antimicrobial Resistance

Antimicrobial resistance (AMR) is the ability of microorganisms to

conform and adapt to antimicrobial medications thereby rendering it ineffective.

AMR is primarily the result of bacterial adaptation to frequent and unsupervised

antibiotic exposure over the years (Katzung, 2018). Microorganisms that develop

antimicrobial resistance are sometimes referred to as “superbugs”. The

biochemical resistance mechanisms used by bacteria include the following:

antibiotic inactivation, target site modification, altered permeability, and “bypass”

of metabolic pathway (Kapoor et al., 2017).

 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 15

Antimicrobial resistance in bacterial infections is a growing global health

concern (Prestinaci et al., 2015). A statement released in February 2018 by the

World Health Organization’s (WHO) confirms that there is a serious situation of

antibiotic resistance worldwide; in particular, according to WHO’s new Global

Antimicrobial Surveillance System (GLASS), there is a widespread occurrence of

antibiotic resistance among 500,000 people with suspected bacterial infections

across 22 countries with some of the world’s most common – and potentially most

dangerous – infections becoming drug-resistant, and cited Staphylococcus aureus

as one of the most commonly reported resistant bacteria (Philippine News

Agency, 2018).

2.3 Methicillin Resistant Staphylococcus aureus

MRSA infections, while for the most part non-threatening, appearing

primarily as skin and soft tissue infections (SSTIs), have substantial morbidity

and mortality (Haysom, 2018). The emergence of new resistance mechanisms

against antimicrobials and the limitations of currently approved treatments makes

it harder to treat infections caused by MRSA resulting in prolonged illness,

serious morbidity such as pneumonia, and death (World Health Organization,

2018); therefore, there is a need to identify alternative agents for the treatment of

MRSA bacteremia (Hassoun et al., 2017).

 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 16

MRSA originated from Staphylococcus aureus (S. aureus) which is a

Gram positive, cocci shaped bacteria. S. aureus is a commensal and an

opportunistic pathogen, and is typically one of the common causes of SSTIs.

SSTIs are defined as an inflammatory invasion of the epidermis, dermis and

subcutaneous tissues (Mishra & Palo, 2016). S. aureus becomes MRSA via the

acquisition of a mobile genetic element carrying the gene encoding resistance to

most beta-lactam antibiotics. It can spread through direct skin-skin contact, or

skin-fomite from an infected or colonized individual, within health care and

within the community (Semret & Haraoui, 2019). A multifactorial range of

independent risk factors for MRSA have been reported in literature which

includes immunosuppression, hemodialysis, peripheral malperfusion, advanced

age, inadequacy of antimicrobial therapy, insulin-requiring diabetes, and

decubitus ulcers, among others (Garoy et al., 2019). MRSA infection is normally

treated with the antibiotic mupirocin (pseudomonic acid A) which is a competitive

inhibitor of bacterial isoleucyl-tRNA synthetase and is active against most ‘Gram-

positive’ and some ‘Gram-negative’ bacilli (Kim & Kwon, 2016). Mupirocin-

mediated inhibition of isoleucyl-tRNA synthetase impedes protein and RNA

synthesis, ultimately leading to bacterial death; however, like any other

antimicrobial agents, it has impediments including slow bactericidal activity, low

tissue penetration, and increasing reports of resistance (Poovelikunnel et al.,

2015).
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 17

2.4 Topical Formulation

Skin is the most readily accessible organ on the human body for topical

administration and is the main route of topical drug delivery system. (Bhowmik

et al., 2012) It is also the most susceptible organ for MRSA infections since it is

spread by skin contact with an infected person, and by sharing personal items that

came in contact with an infected skin. Skin and soft tissue infections (SSTIs) are

the most frequently reported clinical manifestations of MRSA, specifically

furuncles, carbuncles, and abscesses that would eventually become deep and

painful and would require surgical draining. (Center for Disease Control, 2019).

Water removable bases are oil in water emulsions, which are commonly

referred to as creams. This type of ointment base can absorb serous discharges,

and are easily washed from the skin since its external phase is aqueous (Ansel &

Allen, 2014). Creams would be preferred over ointments as it is easily spread

compared to the latter, and would be ideal for the types of wounds that MRSA

inflicts. Compared to other types of bases, creams are not greasy, water washable,

easily spread, and can contain a certain amount of aqueous solution (Ansel &

Allen, 2014).

2.4 Stability Test

 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 18

Stability studies, considered the most important step during the

developmental stages, evaluate the effects of environmental factors on the quality

of the drug substance and determines the stability of the drug product. Stability is

expressed as the shelf life (t90), which is the time necessary for the drug to decay

to 90% of its original concentration therefore losing its quality, safety and efficacy

(Arunachalam & Shankar, 2013; Punam, 2014). The primary reason for

performing such tests is to prevent the patient from taking an unstable drug

product, which can degrade into toxic decomposition products leading to failure

of the therapy, or worse, death (Pokharana, 2018). Conducting stability tests has

become a legal requirement before approval of a new drug product (Bajaj, 2012).

Topical preparations should be evaluated for appearance, clarity, colour,

homogeneity, odour, pH,, consistency, viscosity, assay, preservative and

antioxidant content (if present), microbial limits/sterility and weight loss (ASEAN

Guideline on Stability Study of Drug Product, 2018).

In an accelerated stability testing, a drug product is subjected to several

stress conditions that would accelerate its degradation including: temperature,

moisture, light, agitation, gravity, and pH adjustment (Punam, 2014). The stability

of the drug formulation would be compared to the relative stability of similar

alternative formulations, shortening the time needed to develop a drug

(Pokharana, 2018).

2.5 Research Questions

 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 19

This study aims to formulate an antimicrobial ointment that will be

effective against MRSA strains using the ethanolic extract of Piper betle;

Furthermore, it aims to answer the following questions:

2.2.1 What are the physicochemical properties of the Piper betle ethanolic

leaf extract?

2.2.2 What should be the composition of the Piper betle cream

formulation?

2.2.3 What are the physico-chemical properties of the cream formulation?

2.2.4 Will the formulated Piper betle cream exhibit an antimicrobial effect

against MRSA strains, if so, how different is its antimicrobial effect

compared to the standard mupirocin cream?

2.2.5 How long will the formulated Piper betle cream’s organoleptic

properties, pH value and viscosity remain stable in an accelerated studies

environment?
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 20

CHAPTER 3

THE RESEARCH METHODS

This chapter discusses the research design of the study. It includes the

materials and methods that will be used and the analysis of the gathered results.

3.1 Research Design

The study primarily focuses on the formulation and evaluation of Piper

betle cream and its antimicrobial effect against MRSA. It begins with the

collection and authentication of Piper betle plant. The plant extract, which will be

obtained using the Soxhlet apparatus, will be tested for its different

physicochemical properties. The Piper betle cream will be compounded, followed

by the evaluation of its physicochemical properties, antimicrobial activity and

stability. The details of the different tests utilized shall be discussed in detail in

this chapter. The data gathered will be statistically analyzed. The flow of the

research is shown in figure 1.

 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 21

Figure 1. Research Diagram

3.2 Methods and Materials

3.2.1 Plant Material

The leaves of Piper betle will be obtained from Inarawan, Antipolo City.

The samples will be submitted to the University of Santo Tomas Herbarium

 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 22

Research Section of the Research Center for Natural and Applied Sciences at the

Thomas Aquinas Research Complex (España, Manila, Philippines) for

authentication.

3.2.2 Preparation of Piper betle ethanolic leaf extract

Preparation of ethanolic leaf extract of Piper betle will be done according

to the method presented by Datta and colleagues (2011):

The collected leaves will be air dried and subsequently grinded into

powder. The grounded or powdered leaves will be placed in a Soxhlet apparatus

along with 300mL of 95% ethanol. The Soxhlet apparatus will then be left to run

for 16 hours. The crude plant extract will be removed from the Soxhlet apparatus

and will be concentrated to dryness in a rotary vacuum evaporator at a

temperature below 50 oC. The dried crude plant extract will then be stored in a

refrigerator with 4oC.

3.2.3 Organoleptic Properties of Piper betle leaf extract

Place the extract in a transparent bottle over a white background and

observed the color and clarity. Determine the odor by sniffing and to determine its

characteristic feel to the touch, rubbed between fingers.

3.2.4 Phytochemical Analysis

3.2.4.1 Test for Eugenol

 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 23

Thin layer chromatography will be used to determine presence of eugenol.

5.0 µL of eugenol standard will be dissolved in 10 mL ethanol to produce 0.5

µL/mL solution. HPTLC Silica gel, with the dimensions of 10 x 10 cm, will be

the stationary phase. Whereas, ethyl acetate will be the mobile phase. 5 µL of the

eugenol standard and Piper betle ethanolic extract will be applied, both having

6mm as a band. The plate will be air dried where 8cm will be the developing

distance. Afterwards, the TLC plate will be placed under the following conditions:

a) UV 254 nm; b) UV 366 nm before spraying and; c) Visible light after spraying

with vanillin-sulphuric acid reagent for detection of eugenol (Globinmed, 2019).

3.2.5 Preparation of Piper betle cream

Listed below are for the formulation of an o/w emulsion. Cetyl alcohol

will serve as a thickening agent and stabilizer. Propylene glycol will be the

humectant to reduce the loss of moisture, Span and Tween as emulsifiers.

Methylparaben and Propylparaben will be the preservatives.

Methylparaben, propylparaben, propylene glycol, and tween 80 will be

heated at 70 °C and mixed in water until it becomes homogeneous. Span 80 will

be melted with cetyl alcohol in mineral oil at 70 °C; the two phases will be mixed

under the same temperature. The Piper betle extract will be added at 40 °C after

the two phases are mixed. The mixture will be mixed until it congeals. The

resulting cream will be placed in 50 g amber bottle.

Table 4. Formulation of Piper betle cream

 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 24

Ingredients
I (% w/w) II (%w/w) III (%w/w)

Tween 80 3.731 3.731 3.731

Propylene glycol 3 4 5

Methylparaben 0.18 0.18 0.18

Span 80 3.269 3.269 3.269

Cetyl alcohol 8 9 10

Mineral Oil 20 20 20

Propylparaben 0.2 0.2 0.2

Piper betle extract 15.6 15.6 15.6

Water Qs ad 100 Qs ad 100 Qs ad 100

3.2.6 Physico-chemical evaluation of the Piper betle cream

The formulated Piper betle cream will be evaluated for its organoleptic

characteristics, pH value, viscosity, spreadability, and phase separation.

3.2.6.1 Organoleptic characteristics

The organoleptic properties of the Piper betle cream will be evaluated by

two of the researchers (Patil et al., 2012). The following will be assessed: odor,

physical appearance, texture and homogeneity (Budiman et al., 2018).

 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 25

The cream’s odor will be described according to the 7 primary odors stated

in the stereochemical theory of odor (Ohira, 2007): camphoraceous (cough

medicines like Vicks vaporub), musky (woody scents like sandalwood), floral

(like lavender or rose petals), pepperminty (like peppermint), ethereal (like ether

or cleaning fluid), pungent (like vinegar) or putrid (like rotten eggs).

The cream’s color and consistency (thickness and firmness) will be used to

describe its physical appearance (Patil et al., 2012). Texture will be determined by

how it feels on the skin (stiffness, greasiness, and grittiness); texture should be

smooth so it can easily be spread and penetrate through the skin. The presence of

coarse particles shall be used to evaluate the homogeneity of the formulation. A

homogenous formulation is marked by the absence of coarse particles (Metha et

al, 2013; Chen et al., 2016).

3.2.6.2 pH Value Determination

The pH of the Piper betle cream will be determined by placing 1g of the

cream in 10 mL distilled water which will be allowed to stand for 30 minutes; it

will be subsequently measured using a pH meter (Sawant & Tajane, 2016). For

topical preparations, the pH should be in the range of skin pH (4.5-7.0) to avoid

any irritation to the skin (Budiman et al., 2018).

3.2.6.3 Viscosity

The viscosity (cP) of the formulated Piper betle cream will be determined

by using a Brookefield viscometer DV-II. The test sample will be placed in a

clean and dry 250 ml beaker before placing it under the viscometer. The speed
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 26

(rpm) and the spindle size, ranging from S1 (biggest) to S7 (smallest), will be

determined through trial and error (Chen et al., 2016).

3.2.6.4 Spreadability

The spreadability shall be determined by placing a small amount of

sample in between two slides, having a dimension of 20 ×5 cm, which will then

be compressed to a homogenous thickness by placing it under a weight of 100g

for about one minute. The excess of cream will be scraped off. The slides will be

fixed to a stand at a 45 ̊ angle without the slightest disturbance so that only the

lower slides will be held firmly by the clamp, which will let the upper slide to slip

off freely under a weight of 20g. The time required to separate the two slides will

be measured as spreadability. (Sawant & Tajane, 2016). The lesser time taken for

separation of the two slides, the better the spreadability (Shankar et al., 2016).

Spreadability was calculated by following formula:

S=M×L/T

Where, S= Spreadability, M= Weight tied to the upper slide, L= Length of

glass slide, T= Time taken to separate the slides

3.2.6.5 Centrifugal Test

A centrifugal test will be done immediately after the preparation of Piper

betle cream to determine if its two phases will separate. 5g of the formulated

cream will be placed in disposable centrifugal tube and subsequently placed in a

centrifuge The centrifugal tests will run at 25°C and at 5000 rpm for 10 minutes.
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 27

There should be no phase separation for that is a sign of instability (Akthar et al.,

2011).

3.2.7 Test for Antimicrobial Activity

The handling of active MRSA strains requires a biosafety level 2 working

area; therefore, the Piper betle cream will be submitted to the National Institute

of Molecular Biology & Biotechnology (BIOTECH), University of the

Philippines Los Baños for antimicrobial testing against MRSA. The modified

Kirby Bauer Method, where the MRSA organisms shall be cultured on Mueller-

Hinton agar in the presence of filter paper disks impregnated with the formulated

Piper betle cream, will be utilized. The presence or absence of growth around the

disks is an indirect measure of the ability of that compound to inhibit that

organism (Hudzicki, 2007). The positive control would be mupirocin cream while

the negative control is the base of the cream formulation. The test will be done in

triplicate.

3.2.8 Stability Test

The determination of the physical and chemical stability of the Piper betle

cream will be conducted in accordance with the ASEAN Guideline on Stability

Study of Drug Product (2018). The Piper betle cream will be stored at 40 °C with

75% RH. The effect on the cream’s organoleptic properties, pH value,

spreadability and its phase separation will be checked and recorded weekly for
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 28

one month. The data gathered will be compared to the data from the initial

physicochemical evaluation of the cream to determine the changes that occured.

3.3 Statistical Analysis

The value of the significance of formula = 0.00 with significance value of

<0.05 based on the statistical analysis of Anova test. For further analysis, Duncan

test will be done to determine if there was equality between test treatments in

more detail (to determine the effect of the formula on the amount of microbial

decline in further) (Kunsuma et al., 2018).

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