Antifungal Activity of Bangkal (Nauclea orientalis) Stem and Leaves Extracts against Aspergillus niger1

. Antifungal Activity of Bangkal (Nauclea orientalis) Stem and

Leaves Extracts against Aspergillus niger

Jan Rae B. Atienza1

Bon Mar M. Corpuz2
Brylle Firenze Javier3
John Harvey Montemayor4
1 Project Leader, Grade 10 SSC @ NEHS
2 Member, Grade 10 SSC @ NEHS
3 Member, Grade 10 SSC @ NEHS
4 Member, Grade 10 SSC @ NEHS

ABSTRACT

This investigation was done in order to determine the effectiveness of Bangkal

(Nauclea orientalis) stem and leaves extract in inhibiting the growth of Aspergillus niger.
How the different treatments can affect the colonization of the fungus?

The extracts were obtained by separately soaking 30 grams of dried stem and
leaves in 150 ml of 80% ethanol for 48 hours then filtered and the filtrates were allowed
to evaporate manually to obtain pure extracts of Nauclea orientalis stem, and leaves.
Treatment 1 being the pure stem extract, Treatment 2 being the pure leaves extract,
and Treatment 3 being the combined stem and leaves extract while Treatment 4 being
the Itraconazole (positive control) were tested against Aspergillus niger by single plug
method.

Results showed that the pure (stem and leaves) extracts and combined extracts
exhibited significant antifungal effect on the culture media with mean colonization of
0mm. Data Analysis also shows that the different extracts are comparable to the
commercially used antifungal medicine, Itraconazole.

The findings confirmed that the ethanolic extracts of stem, leaves, and combined
stem and leaves of Nauclea orientalis showed antifungal activity against Aspergillus
Niger.

INTRODUCTION
 Antifungal Activity of Bangkal (Nauclea orientalis) Stem and Leaves Extracts against Aspergillus niger2

The Kingdom Fungi plays some important roles on ecological and economic on
organisms. They break down dead organic materials to continue the cycle of nutrients
through ecosystems. Other fungi provide numerous drugs like penicillin, foods, truffles,
and many more. Fungi can also cause plant and animal diseases; in humans, ringworm,
athlete’s foot, and several more diseases. Fungal diseases are extremely hard to treat
because fungi are more chemically and genetically similar to animals than other
organisms (Speer, 2006).
A member of the genus Aspergillus which contains a set of fungi that are usually
considered asexual, although perfect forms (forms that reproduce sexually) have been
found is called Aspergillus niger. Aspergilli are omnipresent in nature. They are
geographically widely distributed, and have been noticed in a wide range of habitats
because they can populate a broad variety of substrates. A. niger is generally found as
a saprophyte growing on dead leaves, kept grain, compost piles, and other
decomposting vegetation. The spores are widespread, and are frequently associated
with organic substance and soil (Corrigan, 2009). A. niger is not one of those that are so
fatal, but can certainly spawn illness and allergic reactions. A. niger is a very
thermotolerant fungus that can thrive in freezing conditions and very hot weather
(Metzger 2008). This kind of fungi has various toxins, some safe and others dangerous
to specific people. The main toxins it contains are malformin C, and ochratoxin A.
Through fermentation, Aspergillus niger can manufacture useful enzymes that
can be used in the creation of corn syrup, Beano, wine and cider (Rajkumar 2008). It
contains toxins that can make people with weak immune systems become very ill and
can occasionally result in death. These toxins can be inhaled by humans, most often
people who work with plants or peat on their surroundings, and can trigger a lung
disease called Aspergillosis, which has contaminated over 300,000 people worldwide
(Keir, 2013).
` One of the countries blessed with plentiful natural wealth is Philippines. These
plants are useful to all the people of the country providing them source of their
fundamental needs and other necessities. Aside from providing, food, plants have a
special role in drug creation. Drugs are frequently obtained from plants and animals
which are very helpful as treatment for human illnesses. Diverse plants have
determined their values as sources of possible drugs (Ballada & Domondon, 2011).
According to Hiromitsu, Mariko, Noriyuki, (2005) a large tree distributed in India
and is indigenous to north Australia is Nauclea orientalis. Some can also be found here
on Philippines. The concoction from seed pulp is used to cure cough, cold, stomach
pain, vomiting and diarrhea. While the leaves and bark can be medicinally used to treat
abdominal pain, animal bites and wounds (Orwa et al., 2009).
Chemicals produced by plants, in strict sense, can be defined as Phytocemicals.
However, the term is generally used to describe chemicals from plants that may affect
health, but are not essential nutrients. While there is ample evidence to support the
health benefits of diets rich in fruit, vegetables, legumes, whole grains, and nuts,
evidence that these effects are due to specific nutrients or phytochemicals is limited.
Because plant-based foods are complex mixtures of bioactive compounds, information
 Antifungal Activity of Bangkal (Nauclea orientalis) Stem and Leaves Extracts against Aspergillus niger3

on the potential health effects of individual phytochemicals is linked to information on

the health effects of foods that contain those phytochemical (lpi.oregonstate.edu).
In the study of Raghavamma and Rama Rao (2010), the extract from Nauclea
orientalis Linn. leaves show dose-dependent action and inhibition of spontaneous
motility (paralysis) and death of earthworms. This indicates that this plant has
antihilmentic properties. Moreover, phytochemical evaluation of the ethanolic leaf
extract showed the presence flavonoids, phenols, phytosterols and tannins. From the
percentage inhibition calculated the ethanolic extract possesses antioxidant properties
Thusyanthan (2014).
A group of plant metabolites thought to provide health benefits through cell
signalling pathways and antioxidant effects is called Flavonoids. These molecules are
found in a variety of leaves and vegetables (Robertson, 2014). Flavonoids may help
provide protection against diseases like cancer, aging, atherosclerosis, ischemic injury,
inflammation and neurodegenerative diseases (Parkinson’s and Alzheimer’s) by
contributing, along with antioxidant vitamins and enzymes, to the total antioxidant
defense system of the human body (Micronutrient Information Center, 2008).
Any of a gathering of undefined glycosides of terpenes and steroids, happening
in many plants, described by a capacity to shape emulsions and to froth in fluid
arrangements, and utilized as cleansers is called saponin (Dictionary.com). While a
group of pale-yellow to light-brown amorphous substances in the form of powder, flakes,
or a spongy mass, widely distributed in plants and used chiefly in tanning leather,
dyeing fabric, making ink, and in various medical applications is called Tannin also
called as Tannin acid .Tannin solutions are acid and have an astringent (Eugster, n.d.).
Huge and complex particles that assume numerous basic parts in the body are
proteins. They do the vast majority of the work in cells and are required for the
structure, capacity, and direction of the body's tissues and organs. Proteins are
comprised of hundreds or thousands of littler units called amino acids, which are
connected to each other in long chains. There are 20 distinct sorts of amino acids that
can be joined to make a protein (ghr.nlm.nih.gov).
A gathering of atoms with a generally vast episode in nature around the globe
are called alkaloids. They are extremely changed chemicals and biomolecules, yet they
are all auxiliary mixes and are gotten from amino acids or from the transamination
procedure. Alkaloids are ordered by the amino acids that give their nitrogen particle and
some portion of their skeleton. Comparative alkaloids can have very unique biosynthetic
pathways and distinctive bioimpacts. Alkaloids are gotten from L-lysine, L-ornithine, L-
tyrosine, L-tryptophan,L-histidine, L-phenylalanine, nicotinic corrosive, anthranilic
corrosive, acetic acid derivation, or amination and transamination responses. The
terpenoid, steroid, and purine alkaloids are additionally essential. A great many
individuals around the world utilize purine alkaloids consistently whether beginning the
day with some espresso or drinking some tea toward the evening. Alkaloids likewise
happen in the set of all animals. Unlike plants, the source of these molecules in an
animal’s body can be endogenous or exogenous. Alkaloids are particles taking an
interest in both maker and purchaser chains in nature (Aniszewski, 2015)
 Antifungal Activity of Bangkal (Nauclea orientalis) Stem and Leaves Extracts against Aspergillus niger4

A substance that the body creates actually or that can be made as a medication
to treat ailment and wounds is called steroids. Steroids are some of the time utilized
wrongfully by individuals doing games to enhance their execution (Longman Dictionary).
This study entitled “Anti-fungal activity of Bangkal (Nauclea orientalis) stem
and leaves in Aspergillus niger “focuses on finding out the potency of Bangkal stem
and leaves extracts as a substitute for eradicating Aspergillus niger. It aims to: 1.)
Measure the susceptibility of Aspergillus niger to the following treatments in terms of
zone of colonization: (a) 100% stem extract of Nauclea orientalis (b) 100% leaves
extract of Nauclea orientalis (c) 50% stem extract and 50% leaves extract of Nauclea
orientalis 2.) Determine the significant difference among the different treatments. 3.
Determine the significant difference between the antifungal efficacy of Nauclea
orientalis and Itraconazole.

The null hypothesis of this study is that “The stem and fruit extracts of Nauclea
orientalis will not exhibit antifungal activity against Aspergillus niger in terms of zone of
colonization.”

MATERIALS AND METHOD

 Antifungal Activity of Bangkal (Nauclea orientalis) Stem and Leaves Extracts against Aspergillus niger5

Collection of Materials

The following materials were gathered for the conduction of this investigatory
project: mortar and pestle, blender, 240 ml of 95% ethanol, 60 ml of distilled water,
and 30 grams of Nauclea orientalis stem and leaves.

Preparation of Nauclea orientalis Stem and Leaves

The collected stem and leaves were air-dried for four weeks in order to reduce
damage from mould and other microbial infestation (Youngken, 2003). Mortar, pestle,
and blender were used for crushing the dried Nauclea orientalis stems and leaves.

Extraction of Nauclea orientalis Stem and Leaves

The 95% Ethanol was diluted to 80% by adding 20mL distilled water every 80mL
of the Ethanol then 30 grams of Nauclea orientalis stems and leaves were soaked in
separate 150ml 80% ethanol in a vial for 48 hours. The extracts with ethanol were
filtered to remove the solid residues. The filtrates were subjected to manual evaporation
process to remove the presence of alcohol and obtain the pure extracts.

TREATMENTS
T1 100% Stem extract
T2 100% Leaves extract
50% Stem extract
T3
50% Leaves extract
T4(+) Itraconazole
Table 1. Different Treatments

Anti-fungal Screening through In-Vitro Bioassay Testing

Preparation of Test Organism and Media

Pure culture of Aspergillus niger was obtained from the Microbial Culture
Collection and Testing Laboratory, Central Luzon State University. In this study, eight
petri dishes and the PDA were sterilized in an autoclave at 15 psi for 15 minutes. The
 Antifungal Activity of Bangkal (Nauclea orientalis) Stem and Leaves Extracts against Aspergillus niger6

PDA was poured in eight petri plates until in covers the whole area of plate. After the
solidification of PDA, each treatment was pipetted and spread evenly into separate petri
dishes. Each treatment was replicated twice to ensure the reliability of the results.

Protectant test against Aspergillus niger

A plug of fungi Aspergillus niger was placed in the middle of impregnated petri
dish. The growth in media was determined and measured at the nearest mm using a
ruler after 24 hours of incubation. If the media shows no growth of fungi then the
treatment proved to be effective and has inhibiting property against the test organism
(Breslin, et al., 2011). Ruler was used in measuring the diameter of zones of
colonization of the treatments.

Statistical Design and Analysis

The experiments were laid out using the Analysis of Variance (ANOVA). This tool
was used in order to determine if there is a significant difference among the different
treatments used, in terms of zone of colonization of Aspergillus niger.

RESULTS AND DISCUSSION

The effectiveness of the Nauclea orientalis leaves and stem was obtained by
measuring the zone of colonization of the treatment extracts against Aspergillus niger.
The obtained data gives initial results of the in-vitro to determine whether treatments
have active component to control the growth of the fungi.
 Antifungal Activity of Bangkal (Nauclea orientalis) Stem and Leaves Extracts against Aspergillus niger7

The zone of colonization of the Aspergillus niger was determined by measuring

the growth of the initial plug. After 24 hours of of incubation, the following data was
obtained:

Treatments Zone of Colonization

(mm)
T1 0
T2 0
T3 0
T4(+) 0
Table 2. Mean of zone of colonization (mm) of the treatments against Aspergillus
niger

Zone of Colonization (mm)

1
0.9
0.8
0.7
0.6
Zone of Colonization (mm)
0.5
0.4
0.3
0.2
0.1
0
T1 (Pure Stem) T2 (Pure Leaves) T3 (Combined) T4(Itraconazole)

Figure 1. The graph shows the efficiency of the different treatments

against A. niger

The figure shows the comparison of the mean zone of colonization of the test
organism to the different treatments. No growth in the media was observed among all
the treatments. This reveals that all the treatments, exhibit a high antifungal activity
because they show no growth in the media.
 Antifungal Activity of Bangkal (Nauclea orientalis) Stem and Leaves Extracts against Aspergillus niger8

Treatment Replicate 1 Replicate 2 Mean (mm)

T1 (Pure Stem) 0 0 0
T2 (Pure Leaves) 0 0 0
T3 (Combined) 0 0 0
Itraconazole 0 0 0
Table 3. Diameter of zone of colonization (mm) of the test organism to
the treatments.

Conclusions

Based on the findings the following conclusions were drawn:

1) It can be gleaned that Aspergillus niger is susceptible to the following extracts

through zone of colonization:
a. 100% stem extract of Nauclea orientalis

b. 100% leaves extract of Nauclea orientalis

c. 50% stem extract and 50% leaves extract of Nauclea orientalis

2) There is a significant difference among the treatments.

3) The antifungal efficacy of Nauclea orientalis extracts are comparable to the

commercially used antifungal medicine, Itraconazole.

Recommendations:

1) Use different concentrations of ethanol in extraction to test the efficacy of

Nauclea orientalis.
2) Use other parts of Nauclea orientalis as an Antifungal agent.
3) Observe the test organism for 72 hours to give it more time to inoculate.

REFERENCES
Aniszewski T. (2015). Chapter 1 – Definition, typology, and occurrence of alkaloids.
Retrieved March 24, 2017, from
http://www.sciencedirect.com/science/article/pii/B9780444594334000018
 Antifungal Activity of Bangkal (Nauclea orientalis) Stem and Leaves Extracts against Aspergillus niger9

Bangkal / Nauclea orientalis / Leichhardt pine : Philippine Medicinal Herbs / Philippine

herbal Medicine. (n.d.). Retrieved March 03, 2016, from
http://www.stuartxchange.org/Bangkal.html
Breslin B., Ferguson J., Byrum D., Foster M., Kelly D., Lyons M., Mosallam B., Perles
G. (2011) Antimicrobial Resistance Learning Site. Microbiology Module,
Retrieved March 24, 2017, from
http://amrls.cvm.msu.edu/microbiology/microbiology-module
Corrigan B. (October 15, 2009). Aspergillus niger. In Encyclopedia of Life. Retrieved
March 17, 2017, from http://eol.org/pages/2920814/details.
Cruz, J.P. (2014). Evaluation Of The Anti – Staphylococcal Activity Of Nauclea
Orientalis Linn. Retrieved March 06, 2016, from
eujournal.org/index.php/esj/article/download/4329/4149
Dr. S.T.V Raghavamma. (May 2011) In vitro antioxidant potential of crude extract from
leaves of Nuclea orientalis Linn. Retrieved March 04, 2016, from
https://www.researchgate.net/publication/278406417_In_vitro_antioxidant_potent
ial_of_crude_extract_from_leaves_of_Nauclea_orientalis_Linn
Hiromitsu T, Mariko K, Noriyuki K. Chemistry of indole alkaloids related to the
corynanthe-type from uncaria, nauclea and mitragyna plants. Curr Org
Chem. 2005;9:1445–64.
Keir, G. J. et al. (2013). Cyclical caspofungin for chronic pulmonary aspergillosis in
sarcoidosis. Thorax, thoraxjnl-2013.
National Library (2017). “What are proteins and what do they do?”. Retrieved March 25,
2017, from https://ghr.nlm.nih.gov/primer/howgeneswork/protein
Metzger, B. (November 2016) Aspergillus niger . In Mushroom Observer. Retrieved
March 24, 2017, from http://mushroomobserver.org/name/show_name/15030?
_js=on&_new=true
Micronutrient Information Center. (n.d.). Retrieved March 03, 2016, from
http://lpi.oregonstate.edu/mic/dietary-factors/phytochemicals
Orwa, et al. (2009). Agroforestry Database. Retrieved March 24, 2017, from
http://www.worldagroforestry.org/treedb/AFTPDFS/Nauclea_orientalis.PDF
Phenol | chemical compound. (n.d.). Retrieved March 03, 2016, from Wade, L. G. (n.d.).
Phenol | chemical compound. Retrieved March 03, 2016, from
http://www.britannica.com/science/phenol
Rajkumar K. (2008). Multidrug resistant tuberculosis co-existing with aspergilloma and
invasive aspergillosis in a 50 year old diabetic woman: a case report. Retrieved
March 24, 2017, from
https://casesjournal.biomedcentral.com/articles/10.1186/1757-1626-1-303
 Antifungal Activity of Bangkal (Nauclea orientalis) Stem and Leaves Extracts against Aspergillus niger10

Reinagel, M. (2013, November 19). What Are Phytosterols? Retrieved March 03, 2016,
from http://www.quickanddirtytips.com/health-fitness/healthy-eating/know-your-
nutrients/what-are-phytosterols
Robertson, S., BSc. (2014, December 02). What are Flavonoids? Retrieved March 03,
2016, from http://www.news-medical.net/health/What-are-Flavonoids.aspx
Saponin. (n.d.). Dictionary.com Unabridged. Retrieved March 24, 2017, from
Dictionary.com website http://www.dictionary.com/browse/saponin
Speer, B. (2006). Introduction to the Fungi. Retrieved March 24, 2017, from
http://www.ucmp.berkeley.edu/fungi/fungi.html
Tannin | biochemistry. (n.d.). Retrieved March 03, 2016, from
http://www.britannica.com/topic/tannin
Thusyanthan J. (2015). Antioxidant Activity of Some Selected Medicinal Plants of
Family Rubiaceae and Acanthaceae Using 1,1- Diphenyl-2-Picrylhydrazyl (Dpph)
Assay. Retrieved March 24, 2017, from
http://www.dlib.pdn.ac.lk/archive/handle1/4904.

APPENDIX
 Antifungal Activity of Bangkal (Nauclea orientalis) Stem and Leaves Extracts against Aspergillus niger11

Photo Documentation

Extracts Potato Dextrose Agar

Autoclave Pouring of PDA into the petri dish

Application of extracts to the petri dish Seeding of plug of Aspergillus niger

 Antifungal Activity of Bangkal (Nauclea orientalis) Stem and Leaves Extracts against Aspergillus niger12

Zone of Colonization of Aspergillus niger to Nauclea orientalis stem extract

Zone of Colonization of Aspergillus niger to Nauclea orientalis leaves extract

Zone of Colonization of Aspergillus niger to Nauclea orientalis combined

stem and leaves extract
 Antifungal Activity of Bangkal (Nauclea orientalis) Stem and Leaves Extracts against Aspergillus niger13

Statistical Analysis

Different Extracts

With Positive Control