Oregano (Plectranthus amboinicus) Crude

Extract as an inhibitor of Aspergillus niger

Co, Katrina Jasmine,*Acyatan, Micah Allia,*Lacap, Stefanie,*Matta, Paula Laine
Student, Manila Science High School, Taft ave. cor. Padre Faura St. Ermita, Manila
Philippines
*email: royalfeline778@gmail.com

Recent studies show that the Plectranthus species is known for its antifungal property and
components like Terpenes and phenols and also the presence of Aspergillus in foodstuff can
lead to deterioration because it synthesize and excrete toxic secondary metabolites known as
Ochratoxins. For extraction of the extract, 1 kg of fresh leaves was used through crude
extraction (maceration method). Kirby-Bauer test (Agar diffusion test) was used for
antifungal screening. This research was successful to completely inhibit Aspergillus niger
upon the result of the disk diffusion assay.

Keywords: Plectranthus species, Ochratoxins, Terpenes and phenols, Crude extraction

(maceration method), Agar Inhibition analysis(Agar diffusion test) or Kirby-Bauer test, Aspergillus

I. INTRODUCTION
It is estimated that the
uncontrolled growth of molds in food
stuff lead to 5-10% loss of food harvest in
the world annually- not to mention the
amount of food that is wasted every single
day because they have reached their
expiry date (Katalinc, 2008). This is more
alarming especially with the foods of
Filipinos like fruits and vegetables such as
grapes, onion and peanuts which have
short shelf lives. With the growing global
awareness about cancer and other diseases
derived from synthetic chemicals, people
make use of alternative substitute for
these fungicides like these possible new
natural antifungal, essential oils extracted
from traditional spices that have become
sources of various feasibility studies
(Santos, 2015).
Essential oils are highly complex
and volatile and their characteristic odor,
originating from the secondary
metabolism of plants, which can be
obtained by various physicochemical
methods and the study of the function of
the essential oils in leaves was not clearly
understand, and well believed that
essential oils are the waste product of
biosynthesis in plants (Camele et
al.,2014). Past studies proved that the use
of various spices increase the shelf life of
these foods, suggesting that some of these
spices would be able to inhibit spoilage
and probably also in pathogenic
microorganisms (Carmo., et al).
 Physicochemical method of
extracting oils from Oregano may vary
depending on the age and plant part, soil
and climate of the region, susceptibility to
pathogens and production method, but it
will have a high content of terpene,
alcohol, aldehydes, ketones, phenols and
esters, which is responsible for the
characteristic flavor and odor of the plant.
(Biondi.,et al., 2011)
Aspergillus niger is described as
composed of saprophytic species and
known for excretion of secondary
metabolites called ochratoxins that can be
found with other fungi in foods such as
grapes, onions and peanut and also
commonly found in contaminant food and
soil. However, it is also considered one of
the most aggressive pathogens to onions
(Allium sativum), which may cause seed
rot, low germination level, seedling death,
stalk rot, and decreased plant growth,
yield and productivity, causing serious
economic losses (Amico et al., 2008).
Inhibiting the growth of Aspergillus niger
sp., formerly known as Aspergillopsis
nigra by applying oregano extract was
successful because it has such as thymol
or carvacrol that is responsible for
antifungal activity (Ellis.,2003).
Fresh leaves was used for Crude
extraction using Maceration method
because fresh leaves have darker oils that
have these antifungal components for
inhibition (Adeel, 2009). For Inhibitory
screening, Kirby-Bauer test is commonly
used. The paper determined the function
of oregano essential oil as an inhibitor to
Aspergillus niger. The significance of the
study is to have a low cost alternative to
fungicide such as Fungicide Parafungus
80 WP to control the damage due to
molds which may have negative effects on
the consumers.
II. METHODOLOGY
Sample collection
The sample of 1kg of fresh oregano leaves
from plants was purchased from a local
plant market. The plants were brought to
the National Museum for species
verification.
Collection of Microorganism of Interest
For culturing the Aspergillus niger,
Department of Science and Technology-
Industrial Technology Development
Institute provided the said fungi for
Inhibition analysis. 1856 pesos was the
cost including the inhibition analysis.
Extraction of Plectranthus amboinicus
oil
1 kg of fresh Oregano leaves was
extracted by DOST Industrial Technology
development Institute (ITDI) through the
process of Crude extraction using
Maceration method with 2.0L of 95%
ethanol. The fresh P. amboinicus was
osterized and soaked in ethanol for 48
hours then was filtered by rotary
evaporator at 60C under vacuum for 2
hours and was further evaporated using
water bath to obtain semi-solid extract. It
produced 1.4 L of Viscous brown extract
and for semi-solid extract 16.0 g was
filtered. The cost was 1566 pesos for this.
Figure 4 Rotary Vaporator used The following are necessary to evaluate
the result:
Antifungal Activity of Crude Extract
against Aspergillus niger In vitro Reactivity Rating:

Disk Diffusion method or Kirby-Bauer 0- NONE (no detectable zone around the
test was used. Five setups with three specimen)
replicates was performed in this research.
The five setups were paper disk with 1-SLIGHT (some malformed or
positive control, 100% Crude extract of degenerated cells under the specimen)
oregano, the disk without the treatment,
with 50% concentration, and the last one 2-MILD (zone limited under the
is with 25% concentration. The five setups specimen)
had potato dextrose agar as culture media.
Aseptic technique was used in the process 3-MODERATE (zone extends 5 to 10mm
of inoculation. (See Table 1.1) beyond specimen)

Table 1.1 Set-Up Used With Three 4-SEVERE (zone extends greater than 10
Replicates mm beyond specimen)
SAMPLE TREATMENT
Aspergillus niger With 100% Inhibitory Activity Rating- (+++)
with Crude concentration complete, (++) Partial, (+) slight, (-)
Extract negative
Aspergillus niger With 50%
with Crude concentration Evaluation of results
Extract
Aspergillus niger With 25% The result is based on the zone of
with Crude concentration inhibition, Inhibitory Activity Rating and
Extract Reactivity of the crude extract against the
Aspergillus niger With Clotrimazole said fungi. (See Table 1.2)
Aspergillus niger No treatment
Antifungal screening

The zone of the inhibition is based on the

clearing around the paper disk with
treatment and calculation of zone of
inhibition, Inhitory activity and the
radioactivity as a result;
Table 1.2- Results In Raw Data

Sample/Contr Total Reacti Inhibito

RESULTS AND DISCUSSION
ol Mean vity ry
Zone of Activit Antifungal Activity of Crude Extract
Inhibitio y
against Aspergillus niger In vitro
n (mm)
Crude Extract 10. 00 2 +++ The three replicates showed that the
of Oregano
crude extract of oregano produced
Leaves (10
mm) complete inhibitory activity (+++) with
Positive 19.73 3 +++ mild reactivity (2) while clotrimazole
Control: produced moderate reactivity against
Clotrimazole Aspergillus niger after a week. Only the
(10 mm) free disk sample had no inhibitory
Negative 0 0 (-) activity. (See Figure 1)
Control:
Sample Free
Disc
25
(10 mm) m
e
Crude Extract 10.00 2 +++ a 20
of Oregano s
u 15
Leaves 50%
r
(10 mm) e 10
Replicate 1
Crude Extract 10.00 2 +++ m
e 5 Replicate 2
of Oregano n
Leaves 25% t 0 Replicate 3
i
(10 mm) n
m

Figure 1 Results in Three Replicates

Figure 2. Replicate 1 Figure 3. Replicate 2 Figure 4. Replicate 3

For the 3 replicates shown in figures, All the concentration of the extract. For more
replicates showed the same results ignoring details (See Table 1.2)

CONCLUSION AND
RECOMMENDATION

The Disk diffusion method showed that the

application of Crude extract inhibited the
Aspergillus niger with 10 mm diameter of
inhibition for the four concentration. The
result may be affected by different factors like
the temperature and well believed that the ACKNOWLEDGEMENTS
extract was not diffused with the agar and
produced only a low inhibitory activity. In Carmelita Manalo, Annabelle V. Briones,
conclusion, the inhibitory effect after Cecille, Eduardo A. Lanto, Eddie Lonto &
applying the extract, this study promotes the Danilo Tandang
potential use of Plectranthus amboinicus
REFERENCES
extract as alternative for fungicide and
possible treatment for Aspergillopsis even Katalinc V. (2008); Percentage of
though the three setups ended up the same uncontrolled growth of molds in
result because it only means that even the foodstuffs annually . Food
concentration is very low, the result will end Chemistry 94(4): p. 550-557
up with strong inhibitory activity. For future
studies, it is aimed to make this study possible Santos T. M (2015); Diseases derived from
for treating diseases ex vivo. synthetic chemicals; The battle
against microbial pathogens. Basic
DOCUMENTATIONS science, technological advances and
educational programs Formatex
research centers v. 2
D. Amico(2008); Endophytic fungi; Nutrient broth; Department of Biology;
Mycological research v. 12 p.100- university of Missouri- St. Louis
107

Moraes, Paes and Holanda; obtaining pure
culture of bacteria or fungi
Microbiology Researchespp. 348-
356
F. Nazzarro, F. Fratianni L. De Martino;
Essential oils on pathogenic
microorganisms Pharmaceutical
6(2013) pp. 1451-1474
C.N Bake, S.A Stocker; Comparison of
Agar Inhibition and broth
microdillution testing techniques;
Microbio (1991) pp. 553-558
C. Valgas, S.M De Souza, E.FA; Screening
methods to determine the antifungal
activities ;Microbiopp. 485-490
M.A Pfaller; Determination of fungal
activities against yeast and molds;
bacterial testing from bacterial
testing and need for standardization;
Microbial rev (2004) pp. 268-280
J. melatidias; analysis of growth
characteristics of fungi in different
culture media: microbial (2005)
pp.478-484
Ahmed SA, van de Sande WW, Stevens DA,
Fahal A, van Diepeningen AD,
Menken SB, de Hoog GS.2014.
Revision of agents of black-grain
eumycetoma in the order
Pleosporales.Persoonia. 33: 14
Ahmed SA, Stevens DA, van de Sande WW,
Meis JF, de Hoog GS. 2014.
Roussoella percutanea, a novel
opportunistic pathogen causing
subcutaneous mycoses. Med Mycol
52:689-698.
Desnos-Ollivier M, Bretagne S, Dromer F,
Lortholary O, DannaouiE. 2006.
Molecular identification of black-grain
mycetoma agents. J Clin Microbiol.
44: 5
Ahmed SA, de Hoog GS, Stevens Da, Fahal
AH, van de Sande WWJ. 2015. In
vitro antifungal susceptibility of
coelomycete agents of black grain
mycetoma to eight antifungals. Med
Mycol 53:295-301

S. arikan; current statues antifungal

susceptibility testing methods;
mycol, (2006) pp. 319-125

Tang, C.W Straton; advanced

techniques in diagnostic
nd
Microbiology, 2 ed. (2013) pp. 937

Crude extraction retrieved from

www.crudeextraction.com