European Online Journal of Natural and Social Sciences 2022; www.european-science.

com
Vol.11, No 1 (s) Special Issue on Education Research in COVID Era: The Future of the Next Generation
ISSN 1805-3602

Evaluation of the Antimicrobial Activity of Ethanolic Leaf Extract of Tabernae-

montana pandacaqui Lam. against Wound-Infecting Pathogens
Klyde Elmarc Galanta1*, Vivien Faye Herrera1, Sheen Gabrielle Santos1, Noel Saguil2, Armin
Coronado3, Witawat Jangiam4 , and Gary Antonio Lirio1, 5*
1
College of Education, Polytechnic University of the Philippines, 1016 Sta. Mesa, Manila,
Philippines; 2Center for Engineering and Technology Research, Research Institute for Science and
Technology, Polytechnic University of the Philippines, 1016 Sta. Mesa, Manila, Philippines;
3
Research Institute for Science and Technology, Polytechnic University of the Philippines, 1016 Sta.
Mesa, Manila, Philippines; 4Department of Chemical Engineering, Faculty of Engineering, Burapha
University, Saensook, Maung, Chonburi 20131, Thailand; 5Center for Natural Sciences and
Resources Research, Research Institute for Science and Technology, Polytechnic University of the
Philippines, 1016 Sta. Mesa, Manila, Philippines
*
E-mail:[email protected]

Abstract
Wound infections caused by antibiotic-resistant microorganisms are widely recognized as a
major public health concern, particularly in developing countries. To combat antibiotic-resistant mi-
croorganisms, it is critical to develop new antimicrobial agents. Local medicinal plants are viewed
as potential candidates for the treatment of microbial infections. Thus, the purpose of this study is to
determine the antimicrobial activity of Tabernaemontana pandacaqui Lam. leaves against wound-
infecting pathogens. The biologically active constituents of the plant were identified using qualita-
tive phytochemical screening and Fourier Transform Infrared (FTIR) analysis of the crude extract.
The study's findings indicated that T. pandacaqui leaf extract was antimicrobial against a variety of
pathogens tested. The highest zone of inhibition was observed against the pathogenic fungus Candi-
da tropicalis at a mean value of 17.73 ± 2.42 mm, followed by Staphylococcus aureus at a mean
value of 14.73 ± 3.17 mm. However, leaf extracts had no inhibitory effect on Pseudomonas aerugi-
nosa, Methicillin-resistant Staphylococcus aureus (MRSA), and metallo-beta-lactamase Pseudomo-
nas aeruginosa (MβL- P.aeruginosa). The bioactivity of leaf extracts is attributed to the active con-
stituents contained within. The results indicated a statistically significant difference in the leaf ex-
tract's efficacy against Staphylococcus aureus (p=0.007) and Candida tropicalis (p=0.008). The cur-
rent findings indicate that T. pandacaqui leaf extracts have antimicrobial activity against wound-
infecting pathogens. Additional research is needed to determine the antimicrobial potential of the
crude extracts against other infectious agents.
Keywords: antibiotic-resistance, antimicrobial activity, disc diffusion assay, natural prod-
ucts, Tabernaemontana pandacaqui Lam.

Introduction
Over the last few decades, wound infection has remained a significant clinical challenge in
hospitals, particularly in developing countries where adequate health care delivery is hampered by
resource constraints (Kihla et al., 2014). Barbosa and Martins (2018) define wound infection as the
presence of replicating microorganisms within a wound that results in host injury. Generally, it is
caused by a variety of microscopic organisms, including bacteria and fungi. According to Reddy et
al. (2008), open wounds are susceptible to bacterial, fungal, and viral infections and act as an entry
point for foundational infections, putting them at a higher risk for severe complications (Mummed et
al., 2018; Tiwari et al., 2012). Infected wounds frequently produce noxious exudates and toxins, as

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 Klyde Elmarc Galanta, Vivien Faye Herrera, Sheen Gabrielle Santos, Noel Saguil, Armin Coronado,
Witawat Jangiam, and Gary Antonio Lirio

well as the death of regenerating cells (Flanagan, 2003; Jalalpure et al., 2008). To alleviate discom-
fort and pain associated with wounds, to prevent infection, and to activate tissue repair processes, it
is necessary to promote wound healing and restore normal body functions to the affected area (Red-
dy et al., 2008).
Each year, approximately 3,300 people die directly as a result of an infection caused by anti-
biotic-resistant microorganisms, and the risk of such infections is comparable to that of infectious
diseases such as influenza, tuberculosis, and HIV/AIDS combined (Cassini et al., 2019). Additional-
ly, a World Health Organization-funded survey discovered a prevalence of nosocomial infections
ranging from 3 to 21%, with wound infections accounting for 5 to 34 %. Wound infection was one
of the top five causes of morbidity in the Philippines. This was confirmed by a study conducted at
the University of the Philippines-Philippine General Hospital. According to the findings, wound in-
fection is a significant cause of morbidity and mortality in patients, owing to the steady increase in
antibiotic-resistant microorganisms (Abesamis & Cruz, 2019). Antibiotic-resistant microorganisms
pose an alarming and growing threat to modern health care.
Antibiotic-resistant microorganisms are rapidly spreading across the globe, jeopardizing an-
tibiotic efficacy (Golkar et al., 2014). As demonstrated by Lomazzi et al. (2019), microorganisms
such as bacteria, fungi, and viruses that are highly exposed to antimicrobial drugs can develop the
ability to resist the drugs intended to eradicate them, jeopardizing antibiotics' ability to treat com-
mon diseases. Mummed et al. (2018) discovered that bacteria and fungi such as Staphylococcus au-
reus, Escherichia coli, Pseudomonas aeruginosa, Candida spp., and Aspergillus spp. are frequently
associated with wound infections, and several of them are antibiotic-resistant. These pathogens have
the potential to significantly slow, if not completely halt, the wound healing process, resulting in
functional limitations of the damaged tissue, which can exacerbate pain and discomfort (Annan &
Houghton, 2008). Indeed, the emergence of antibiotic-resistant pathogens has grown to be a signifi-
cant global threat. This highlights the critical need for new antibiotics to treat common infections
such as wound infection.
Over the last few years, there has been an increase in the development of novel antibiotics
capable of combating the global threat of antibiotic resistance. Local medicinal plants are being
screened for antimicrobial activity and are being considered as promising candidates (Dan et al.,
2018). Medicinal plants have been critical to human health and well-being since the dawn of civili-
zation (Bag et al., 2012). They are typically made with natural ingredients and are used to treat mi-
nor or chronic ailments. Natural products are small molecules that biological organisms synthesize
(Krause & Tobi, 2013). Numerous studies have concluded that natural products are more affordable,
environmentally sustainable, and less toxic than commercially available antibiotics or have fewer
side effects (Lirio et al., 2019; Moloney, 2016). The World Health Organization estimates that ap-
proximately 80% of the world's population lives in developing countries and is heavily reliant on
medicinal plants for health care due to cultural traditions or a lack of alternatives. Plants contain an
abundance of bioactive compounds with antimicrobial properties that can be used to treat or prevent
microbial infections in a cost-effective and safe manner (Bag et al., 2012). Thus, it is critical to con-
duct scientific trials to determine the efficacy of indigenous medicinal plants in order to develop
new and effective antimicrobial agents.
In the Philippines, the Department of Health-Philippine Institute of Traditional and Alterna-
tive Health Care has endorsed only ten medicinal plants following scientific validation to ensure
their safety and efficacy (Boy et al., 2018; Principe & Jose, 2002). Although a number of medicinal
plants in the Philippines have demonstrated promising potential for wound healing, many remain
untested and their use is either poorly regulated or not regulated at all. One such plant is Tabernae-

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Special Issue on Education Research in COVID Era: The Future of the Next Generation

montana pandacaqui Lam., commonly referred to as pandakaki-puti in the Philippines, is a member

of the dogbane family, Apocynaceae. Members of this family, as Gruyal and Medina (2019) note,
are exploited for their secondary metabolites, most notably alkaloids. Secondary metabolites have
been shown to have a variety of biological effects, establishing a scientific basis for the use of herbs
in traditional medicine, as well as possessing antimicrobial properties (Hussein & El-Anssary,
2019). Numerous studies on T. pandacaqui have been conducted, and the results and findings indi-
cate that this species possesses antiprotozoal (Bradacs et al., 2011), anti-inflammatory, antipyretic,
and antinociceptive (Taesotikul et al., 2003), cardiovascular effects (Taesotikul et al., 1989), anti-
bacterial (Gruyal et al., 2019), and antihelminthic properties (Mandanas, 2018). Apart from the stu-
dies mentioned, limited attempts were made to explore the antimicrobial properties of T. pandacaqui
leaves.
T. pandacaqui's various parts have been used medicinally in the past. There are, however, a
few scientific studies that have evaluated the antimicrobial activity of T. pandacaqui leaves against
wound-infecting pathogens. Thus, the purpose of this study is to examine the antimicrobial potential
of ethanolic leaf extract of T. pandacaqui against common pathogens associated with wound infec-
tion and to serve as a natural source to supplement the growing need for effective antibiotics to
combat the problem of antibiotic-resistant microorganisms.

Materials and Methods

Specimen Collection Plant Material
The leaves of T. pandacaqui were chosen for investigation of its antimicrobial properties.
Morphologically, T. pandacaqui leaves are elliptical to narrowly elliptical in shape, as described by
Leeuwenberg (1988). The apex is either obtuse or occasionally rounded. The secondary veins are
usually pale green on both sides, and the petiole measures between 2 and 20 mm (Figure 1).
Specimen Collection, Preparation, and Identification
Fresh leaves of T. pandacaqui were collected in Brgy. San Eugenio, Natividad, Pangasinan,
Region I, Philippines (latitude 16.0382°N, longitude 120.8384°E). The sample was placed in an air-
tight polypropylene-lock bag with the appropriate labeling and washed carefully with clean water to
remove any dust, dirt, residue, or other contaminants. Until used, the cleaned leaves were stored and
air-dried in a cool, dry location. To ensure proper identification, plant specimens were authenticated
at the Institute of Biology Jose Vera Santos Memorial Herbarium (PUH), College of Science, Uni-
versity of the Philippines-Diliman (UPD), Diliman, Quezon City, Philippines.
Extraction of Plant Sample
The leaves of T. pandacaqui were dried for approximately 14 days at room temperature us-
ing shade drying (Chao et al., 2017). These dried samples were cut and crushed in an electric blend-
er until a 90-gram fine powder was obtained. The fine powder was collected into a clean vessel and
soaked in 95% ethanol at a ratio of 1:3 w/v (Mailoa, 2013), in which 270 mL of ethanol was added
to 90 grams of plant fine powder and stored for three days with occasional shaking (Gupta et al.,
2011). Following maceration, the homogenates were filtered through a filter No. 39 (Dent et al.,
2013) and transferred to a 200 mL beaker. Excess solvent was evaporated from the filtered extracts
by placing them in a water bath at a low temperature for more than an hour (Gupta & Dhawan,
2016). After the evaporation process was complete, the sample was scraped, collected, and trans-
ferred to a small glass tube, from which a 12 mL crude extract was obtained and stored aseptically in
a laboratory refrigerator for subsequent use.

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 Klyde Elmarc Galanta, Vivien Faye Herrera, Sheen Gabrielle Santos, Noel Saguil, Armin Coronado,
Witawat Jangiam, and Gary Antonio Lirio

(a) (b)

(c)

(d)

Figure 1. Tabernaemontana pandacaqui Lam. leaf external morphology: (a) elliptical shape,
(b) obtuse apex, (c) pale green secondary veins, and (d) short petiole.

Test Microorganisms
The antimicrobial activity of T. pandacaqui leaves was evaluated against the common
wound pathogens, including two multidrug-resistant bacteria, one gram-positive and negative bacte-
ria, and one pathogenic fungus. They were selected on the basis of their clinical and pharmacologi-
cal significance. The bacterial and fungal strains were provided by the Polytechnic University of the
Philippines' Microbial Collection Culture (PUPMCC). While, the multidrug-resistant strains were
obtained from the De La Salle University's Microbial BioBanks (DLSU-MB). Table 1 lists the pa-
thogens and resistant phenotypes used in the study.

Table 1. Selected pathogens and resistant phenotypes used

Pathogens Resistant Phenotypes
Multi- drug resistant bacteria
MR- Staphylococcus aureus
MβL- Pseudomonas aeruginosa
Gram-positive bacteria Trimethoprim-sulfamethoxazole,
Staphylococcus aureus Cefoxitin, Oxacillin, Penicillin,
(BIOTECH 1582) Amikacin, Cefepime, Ceftadizime,
Gram-negative bacteria Imipenem, Meropenem
Pseudomonas aeruginosa (BIOTECH 1335)
Fungus
Candida tropicalis (BIOTECH 2085)

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Special Issue on Education Research in COVID Era: The Future of the Next Generation

Phytochemical Analysis
Following standard procedures, the ethanolic leaf extract of T. pandacaqui was subjected to
qualitative phytochemical analysis to determine the presence and absence of biological constituents
such as flavonoids, alkaloids, tannins, glycosides, saponins, resins, phytosterols, anthraquinones,
carbohydrates, reducing sugars, and proteins with peptide bonds. The analysis was conducted at the
National Institutes of Health's Institute of Pharmaceutical Sciences at the University of the Philip-
pines Manila (UPM) in Manila, Philippines. Table 2 summarizes the test results.

Table 2. Qualitative phytochemical screening procedures and its positive indicator

Tests Positive Indicator
For Carbohydrates
Molisch Test Violet ring at the junction
For Reducing Sugars
Fehling’s Test Formation of brick red precipitate
For Flavonoids
Alkaline Reagent Test Yellow coloration which disappears upon the addition
of dilute acid
Lead Acetate Test Presence of yellow turbidity or precipitate
For Alkaloids
Hager’s Test Yellow precipitate or turbid solution
Mayer’s Test White precipitate or turbid solution
Wagner’s Test Reddish brown or turbid solution
For Tannins
Ferric Chloride Test Blue solution- presence of gallic tannins
Green to black solution- presence of catecholic tannins
For Glycosides
Keller Killani Test Reddish brown/purple ring at the junction
For Saponins
Froth Test Froth greater than 2 cm even after 30 seconds
For Resins
Test for Resins Turbid Solution
For Phytosterols
Liebermann-Burchard Test Brown ring at the junction
Green upper layer- presence of sterols
Deep red- presence of triterpenoids
For Anthraquinone
Test for Anthraquinone Red color
For Proteins (Peptide Bonds)
Test for Resins Purple violet color

Fourier Transform Infrared Spectroscopy (FTIR) Analysis

FTIR analysis was performed on the ethanolic leaf extract of T. pandacaqui to determine the
functional groups and types of chemical bonds present in the crude extract. The analysis was con-

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 Klyde Elmarc Galanta, Vivien Faye Herrera, Sheen Gabrielle Santos, Noel Saguil, Armin Coronado,
Witawat Jangiam, and Gary Antonio Lirio

ducted at the University of Santo Tomas-Research Center for Natural and Applied Sciences (UST-
RCNAS), located in Manila, Philippines.
Antimicrobial Assay
The antimicrobial activity of Tabernaemontana pandacaqui Lam. leaf extract was deter-
mined using a modified Kirby- Bauer disk diffusion susceptibility test protocol (Bauer et al., 1966).
In a petri dish, Whatman filter paper discs were placed and autoclaved for 45 minutes (Kala
et al., 2010). The sterile discs were inserted into sterile 96-well microplates and dispensed 100 μl of
crude extracts using a sterile micropipette. The discs were then dried at room temperature. The test
microorganisms prepared by growing the cultures in Mueller-Hinton broth (MHB) and incubated for
24 h, followed by turbidity level adjustment to match 0.5 McFarland standards, or 1.5 108 colony-
forming unit (CFU)/ml. Standardized test organisms were swabbed over the sterile Mueller-
Hinton agar (MHA) plate surface. Using a sterile pointed forceps, the discs containing T. pandaca-
qui leaf extracts were impregnated onto the plate.
The plates were inverted and allowed to set for approximately 1 h and incubated for 24 h.
Antimicrobial activity was determined by observing and measuring the diameter of the zone of inhi-
bition (ZOI) surrounding the discs using a millimeter-scale Vernier caliper. The following standard
antibiotics were used as positive controls for bacteria: Rifampicin (5 mcg/ disc, TM Media), Trime-
thoprim (5 mcg/ disc, TM Media), Penicillin (10 units/ disc, TM Media), and Ofloxacin (5mcg/ disc,
TM Media); Nystatin was used as a antibiotic control for fungi. Meanwhile, the discs containing ste-
rile water served as the negative control. All tests were done in triplicates.
Statistical Analysis
The experimental results were expressed as the mean and standard deviation (SD) of the rep-
licates. To determine significant group differences, mean values were compared between ZOIs of
plant extracts and antibiotic control using one-way analysis of variance (ANOVA) and t-test using
JASP version 0.14.1 (JASP Team 2020). Means were considered statistically significant if p-value is
less than 0.005.

Results and Discussion

Antimicrobial Activity
(a (b

Figure 2. Disc diffusion assay results of ethanolic leaf extract of Tabernaemontana pandacaqui
Lam. against wound-infecting pathogens: (a) leaf extract against Staphylococcus aureus, and
(b) leaf extract against Candida tropicalis.

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The efficacy of Tabernaemontana pandacaqui Lam. leaf extracts against several microor-
ganisms associated with wound infection was determined using the disc diffusion method. Leaf ex-
tracts were found to have varying degrees of antimicrobial activity against test microorganisms. As
illustrated in Figure 2, disc diffusion analysis revealed that T. pandacaqui leaf extract had a signifi-
cant inhibitory effect on gram-positive bacteria and fungi, Staphylococcus aureus and Candida trop-
icalis, respectively. On the other hand, T. pandacaqui Lam. leaf extracts had no inhibitory effect
against gram-negative bacteria, Pseudomonas aeruginosa, and multidrug-resistant bacteria, Staphy-
lococcus aureus (MRSA) and Pseudomonas aeruginosa (MβL).
Five antibiotics were used as positive controls in this study: Rifampicin (5 mcg/ disc), Tri-
methoprim (5 mcg/ disc), Penicillin (10 units/ disc), Ofloxacin (5mcg/ disc), and Nystatin. Table 3
summarizes the antimicrobial activity of T. pandacaqui leaf extracts and antibiotic control against
wound-infecting pathogens. The research findings indicate a statistically significant difference in the
efficacy of the leaf extract compared to a positive control against Staphylococcus aureus (p=0.007)
and Candida tropicalis (p=0.008), as determined by One-Way Analysis of Variance (ANOVA) and
t-test at 0.05.

Table 3. Average diameter of the zone of inhibition of the plant extracts and antibiotic
Treat- Antibiotics Control
ment
Pathogens Leaf ex- Rifampi- Trime- Ofloxacin Penicil- Nystatin P value
tract cin thoprim lin
Staphylococ- 0 18. 43 ± 20. 27 ± 19. 73 ± 19. 83 ± 0 0
cus aureus 0.23 0.46 0.92 1.62
(MRSA)
Pseudomo- 0 0 0 0 0 0 0
nas aerugi-
nosa (MβL)
Staphylococ- 14. 73 ± 15. 17 ± 16.93 ± 17.40 ± 17. 67 ± 0 0.007 S
cus aureus 3.17 0.98 16. 1.44 17. 0.52 3.58
Pseudomo- 0 17. 87 ± 22. 03 ± 18. 07 ± 0 0 0
nas aerugi- 1.04 0.40 19. 0.98
nosa
Candida 17. 73 ± 0 0 0 0 10. 83 ± 0.008 S
tropicalis 2. 42 0.98
Note: S statistically significant p-value (p ≤ 0.05)

In general, the present study's findings indicate that the ethanolic leaf extract of T. pandacaqui
possesses antimicrobial activity against the microorganisms tested. The comparison of the efficacy
of T. pandacaqui leaf extracts and antibiotic control against wound-infecting pathogens is presented
in Figure 3. The ethanolic leaf extract of T. pandacaqui exhibited the highest zone of inhibition
against Candida tropicalis, with a mean value of 17.73 2.42 mm, followed by Staphylococcus au-
reus, with a mean value of 14.73 3.17 mm. However, the leaf extract had no inhibitory effect on any
of the other microorganisms tested.

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 Klyde Elmarc Galanta, Vivien Faye Herrera, Sheen Gabrielle Santos, Noel Saguil, Armin Coronado,
Witawat Jangiam, and Gary Antonio Lirio

Figure 3. Comparison of antimicrobial activity of Tabernaemonatana pandacaqui Lam. leaf

extracts and antibiotics control (Rifampicin, Trimethoprim, Ofloxacin, Penicillin, and
Nystatin) against wound infecting pathogens.

The findings of this study are consistent with those of other Tabernaemontana species. Inte-
restingly, Sathishkumar et al. (2012) discovered that the leaves of Tabernaemontana heyneana Wall.
possessed a broad antimicrobial spectrum against microorganisms tested. While Ruttoh et al (2010)
findings indicate that there were significant differences in the inhibitory activity of Tabernaemonta-
na stapfiana between Gram-positive and Gram-negative bacteria, with Gram-positive bacteria exhi-
biting significantly greater inhibitory activity.
The resistance of Gram-negative bacteria may be attributed to the structure of their cell wall.
Numerous studies have demonstrated that the outer membrane of Gram-negative bacteria such as
Pseudomonas aeruginosa can act as an effective permeability barrier against the penetration of toxic
particles such as antibiotics (Choi & Lee, 2019; Tegos et al., 2002). Additionally, several studies
have demonstrated that Gram-positive bacteria are more susceptible, and even exhibit a greater ef-
fect, to antimicrobials derived from plants, than Gram-negative bacteria, which are generally more
resistant due to the presence of the outer membrane (Koohsari et al., 2015; Rameshkumar et al.,
2007; Tajkarimi et al., 2010,). Additionally, Ruttoh et al. (2010) stated that the thick, porous pepti-
doglycan layer within the Gram-positive semipermeable membrane allows for the passage of sub-
stances such as antibiotics and penetration into the cell, making it more susceptible.
Phytochemical Analysis
The presence of secondary metabolites such as flavonoids, alkaloids, tannins, glycosides, sa-
ponins, phytosterol, reducing sugars, and carbohydrates was determined by phytochemical analyses
of T. pandacaqui leaf extract, as summarized in Table 4.

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Table 4.Qualitative phytochemical analysis for the ethanolic leaf extract of T. pandacaqui
Tests Actual Results Indication
For Carbohydrates
Molisch Test Violet ring at the junction Positive
For Reducing Sugars
Fehling’s Test Brick red precipitate Positive
For Flavonoids
Alkaline Reagent Test Light yellow-green solution Positive
Lead Acetate Test Yellow precipitate Positive
For Alkaloids
Hager’s Test Turbid yellow solution Positive
Mayer’s Test Turbid yellow-green solution Positive
Wagner’s Test Turbid reddish-brown solution Positive
For Tannins
Ferric Chloride Test Greenish-black solution Positive
For Glycosides
Keller Killani Test Brown ring at the junction Positive
For Saponins
Froth Test Persistent froth formation Positive
For Resins
Test for Resins Clear yellow solution Negative
For Phytosterols
Liebermann-Burchard Test
Brown ring at the junction Positive
For Anthraquinone
Test for Anthraquinone Clear orange solution Negative
For Proteins (Peptide Bonds)
Test for Resins Greenish-white precipitate Negative

The phytochemicals identified in the extract may explain the inhibitory activity observed
against the bacterial and fungal strains used in the study. According to Shahidi Bonjar (2004), plants
use biological constituents such as flavonoids, alkaloids, tannins, saponins, and a variety of other
secondary metabolites to defend themselves against invasion by a variety of microorganisms, in-
sects, and other herbivores.
Flavonoids are hydroxylated phenolic compounds that possess antimicrobial activity against a
broad spectrum of microorganisms. Flavonoids inhibit the function of the cytoplasmic membrane,
nucleic acid synthesis, energy metabolism, attachment and biofilm formation, alter membrane per-
meability, and decrease pathogenicity (Xie et al., 2014). Flavonoids have been attributed their anti-
microbial activity to their structural functionalities (Daglia et al., 2012). According to Wu et al.
(2013), antimicrobial activity of flavonoids is dependent on their hydroxyl groups. Previously pub-
lished research established a strong correlation between the chemical structure of flavonoids and
their inhibitory activity against pathogens, demonstrating that removing the hydroxyl group from
flavonoids significantly reduces their antimicrobial activity (Sichel, 1991; Tripoli et al., 2007).

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 Klyde Elmarc Galanta, Vivien Faye Herrera, Sheen Gabrielle Santos, Noel Saguil, Armin Coronado,
Witawat Jangiam, and Gary Antonio Lirio

Alkaloids are a diverse group of naturally occurring bioactive compounds found in plants, an-
imals, bacteria, and fungi. This phytochemical played a critical role in the development of new an-
timicrobial agents. Thawabteh et al. (2019) state that the mechanism of action of alkaloid antimi-
crobial agents varies by class. Several classes act as respiratory inhibitors by inhibiting the enzyme
dihydrofolate reductase, thereby inhibiting nucleic acid synthesis; others act as respiratory inhibitors
by decreasing the treated microorganisms' oxygen consumption.
Additionally, tannins were identified in this study. These antimicrobial molecules exert their
effect by inhibiting extracellular microbial enzymes, depriving bacteria of essential growth sub-
strates, or directly affecting microbial metabolism via inhibition of oxidative phosphorylation (Scal-
bert, 1991). Saponins, on the other hand, possess antimicrobial properties due to the presence of a
lipophilic portion (aglycon or sapogenin) and a hydrophilic core composed of one or more sugars
(Costa, 2010). According to Ravi et al. (2016), saponin acts as a chemical barrier in the plant's de-
fense system when pathogens are encountered. This phytochemical may result in the expulsion of
proteins and enzymes from the cell.
Fourier Transform Infrared Spectroscopy Analysis
The FTIR analysis is critical for deciphering the chemical functionality of a compound found
in a plant sample (Prabha et al., 2014). As illustrated in Figure 4 and Table 5, the FTIR analysis of
T. pandacaqui leaf extract revealed the presence of functional groups such as N-O stretching for ni-
trogen compounds (1523.83 cm-1), C=C stretching for alkene and conjugated alkene (1638.60 cm-1),
O=C=O stretching for carbon dioxide (2341.68 cm-1), C-H and N-H stretching for alkane and amine
salt, respectively (2929.03 cm-1), and O-H. The FTIR spectroscopic analysis of T. pandacaqui etha-
nolic leaf extract revealed that major peaks at 3283.95 cm-1 were observed, which could be attri-
buted to the O-H stretching. This indicates that the primary functional group in T. pandacaqui leaf
extract is O-H, which is equivalent to alcohol and carboxylic acid.

Figure 4. The FTIR spectrum analysis of ethanolic leaf extract of T. pandacaqui Lam.

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Special Issue on Education Research in COVID Era: The Future of the Next Generation

Table 5.The FTIR spectral wave number values and functional group obtained from the etha-
nolic leaf extract of T. pandacaqui
Characteristic Absorption (cm-1) Functional Group Bond
1523.83 Nitro compound (Stretch) N-O
1638.60  Alkene (Stretch) C=C
Conjugated Alkene (Stretch)
2341.68 Carbon Dioxide (Stretch) O=C=O
2929.03 Alkane (Stretch) C-H
Amine Salt (Stretch) N-H
3283.95 Alcohol (Stretch) O-H
Carboxylic Acid (Stretch)

The FTIR spectroscopic analysis of ethanolic leaf extract of T. pandacaqui revealed that ma-
jor peaks were observed at 3283.95 cm−1 which could be assigned to the O-H stretching. This indi-
cates that the primary functional group present in T. pandacaqui leaf extract is O-H corresponding
to alcohol and carboxylic acid.
The present study on plant samples revealed the presence of not only biologically active con-
stituents, but also a variety of functional groups that contributed significantly to antibiotic action.
Clearly, FTIR analysis of T. pandacaqui ethanolic leaf extract revealed the presence of nitro com-
pounds, alkenes, conjugated alkenes, carbon dioxide, alkanes, amine salts, alcohol, and carboxylic
acid as a major functional group. According to Burman et al. (2012), the plants' antimicrobial activi-
ty is attributed to functional groups such as alcoholic, aromatic, amine, and carbo-acids found in
tannins, alkaloids, and flavonoids that can be extracted from the plant and used in herbal drug prepa-
rations for the treatment of a variety of bacteria-borne diseases.
The presence of functional groups confers a variety of beneficial properties (Prabha 2014).
The major peak observed in the O-H stretch could be attributed to alcohol or carboxylic acid in this
study. Prasanna & Anuradha (2016) demonstrated that carboxylic acids are critical metabolic prod-
ucts in the formation of fat in the body and act as potent antimicrobial agents. This organic com-
pound was previously used in pharmaceuticals as a primary ingredient in the treatment of a variety
of illnesses, including ulcers, nasal congestion, jaundice, headache, liver pain, edema, and rheumatic
joint pains. Additionally, previous research has demonstrated that metabolites containing alkene,
alcohol, and hydroxyl have significant antimicrobial activity (Chopra & Roberts, 2001). According
to Moovendhan et al. (2014), these functional groups are responsible for cleavage of the bacterial
cell wall and inhibition of cellular respiration and electron transport in their mode of action.
Interestingly, the FTIR analysis results obtained in this study also confirmed the presence of
the detected phytochemical in the plants. For example, the presence of alkaloids is explained by the
N-H stretch at the 2929.03 cm1 position. Meanwhile, the presence of flavonoids is a result of the
peak at 3283.95 cm1, which revealed the presence of the O-H stretch (Jabamalairaj et al., 2015). The
spectroscopic analysis revealed the presence of biologically active functional groups, indicating the
presence of active phytochemicals that may contribute to the plant's bioactivities, such as bactericid-
al and antimicrobial activity.

Conclusions
In the current investigation, the efficacy of ethanolic leaf extract of Tabernaemontana pan-
dacaqui Lam. against several microorganisms associated was evaluated. The findings of this study

Openly accessible at http://www.european-science.com 297

 Klyde Elmarc Galanta, Vivien Faye Herrera, Sheen Gabrielle Santos, Noel Saguil, Armin Coronado,
Witawat Jangiam, and Gary Antonio Lirio

indicate that the ethanolic leaf extracts of T. pandacaqui have significant inhibitory activity against
multidrug-resistant Staphylococcus aureus, and pathogenic fungus, Candida albicans. The plant ex-
tracts have been found to possess a variety of biologically active phytochemicals and functional
groups, all of which have been reported to have inhibitory properties. However, it is necessary to
conduct additional research in order to determine the potential effectiveness of crude extracts as an-
timicrobial agents. The present results will form the basis for selection of plant species for further
investigation in the potential discovery of new natural bioactive compounds.

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