Volume 10, Issue 7, July – 2025 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165 https://doi.org/10.38124/ijisrt/25jul1959

Phytochemical Analysis and Biological

Activity of the Leaf Extracts of Polyalthia
longifolia (Masquerade Tree)
Nzota Joy Chidinma1; Obi Leonard Kelechukwu2*;
Adewumi Chizoma Nwakego3
1;2;3
Department of Pure and Applied Chemistry, Faculty of Natural and Applied Sciences,
Veritas University, Bwari, Abuja, Nigeria

Corresponding Author: Obi Leonard Kelechukwu2*

Publication Date: 2025/08/11

Abstract: The serious global health concern of antimicrobial resistance has prompted the search for new antimicrobials.
Plants are considered a rich source of potent anti-infective agents. This study aimed to assess the phytochemical constituents
of the Nigerian Polyalthia longifolia and its antimicrobial potency, based on its ethno-medicinal use. The crude extract was
obtained by extracting the powdered air-dried leaf of Polyalthia longifolia with methanol. Standard chemical procedures
were used to screen the extract for phytochemicals. Solvent-solvent extraction was used to separate the crude extract into
hexane, neutral, acid, and base fractions. The crude extract and fractions were tested for activity against Salmonella typhi,
Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Candida albicans, and Aspergillus niger. FT-IR and GC-MS
analyses were employed to identify functional groups and specific bioactive compounds in the crude extract. Phytochemical
analysis revealed that terpenoids, alkaloids, saponins, tannins, flavonoids, steroids, and volatile oils were present. The neutral
and base fractions exhibited notable antibacterial efficacy, particularly against Salmonella typhi and Staphylococcus aureus,
with inhibition zones comparable to standard antibiotics. The fungi Aspergillus niger and Candida albicans were not
inhibited by the crude extract or its fractions. Major compounds such as diisooctyl phthalate, oleic acid, palmitic acid,
farnesol formate, n-hexadecanoic acid, octadecanoic acid, and methyl kolavenate were identified by comparing the crude
extract’s GC-MS analysis with reference library computer mass spectrometry data. These compounds may be responsible
for the observed bioactivity. The findings support the traditional use of P. longifolia which is a potential source of
antibacterial agents for novel drugs.

Keywords: Polyalthia longifolia , Leaf Extracts, Phytochemicals, Antimicrobial Activity.

How to Cite: Nzota Joy Chidinma; Obi Leonard Kelechukwu; Adewumi Chizoma Nwakego (2025) Phytochemical Analysis and
Biological Activity of the Leaf Extracts of Polyalthia longifolia (Masquerade Tree).
International Journal of Innovative Science and Research Technology,
10(7), 3434-3442. https://doi.org/10.38124/ijisrt/25jul1959

I. INTRODUCTION
resistance and improve the potency of existing treatments [3].
Antimicrobial resistance (AMR) caused by antibiotic Polyalthia longifolia, also known as “Green Champa” or
misuse and overuse [1] poses a threat to human life, marked “Masquerade Tree” is a tall evergreen tree that is native to
by microorganisms developing resistance to drugs designed India and Sri Lanka. It has been used in traditional medicine
to combat them [2]. The abuse of antibiotics makes resistance to treat helminthiasis, diabetes, fever, skin conditions, and
genes more likely to proliferate, which is gradually increasing hypertension [4]. It has been discovered that the leaves and
and may emerge as the top cause of mortality by 2050, and the roots contain phytosterols and monounsaturated fatty
treating infections can be difficult due to resistant acids, which helps in lowering cholesterol [4]. The plant has
microorganisms [1]. traditionally been used to increase breathing, reduce blood
pressure, stimulate and treat ailments such as uterine
As drug-resistant pathogens and diseases continue to disorders, gonorrhea, leucorrhea, and menstrual issues [5].
emerge, there is a need to investigate medicinal plants According to earlier research, the most prevalent
continuously for possible antimicrobial agents. Medicinal phytochemicals in Polyalthia plants include terpenes and
plants contain bioactive compounds that fight against drug alkaloids [6]. Polyalthia species also contains sterols,

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flavonoids, organic acidssteroids, phytosterols, resins, was then filtered with a filter paper and glass funnel. A rotary
phenols, flavonoids, tannins, steroidal and cardiac glycosides, evaporator set at 40 °C was used to evaporate the filtrate to
and saponins [6,7,8,9]. Methanolic extract of the leaf of dryness. A solid residue with dark green colouration was
Polyalthia longifolia exhibited bactericidal activity against obtained. The crude extracted was spotted on a thin-layer
P. aeruginosa, E. coli, S. pneumoniae, S. aureus and C. chromatography (TLC) plate (an aluminum sheet pre-coated
difficile and fungucidal activity against A. niger, C. albicans, with silica gel) and using 1:5 ethyl acetate/ hexane as mobile
A. clavatus, , A. fumigatus, C. auris and C. tropicalis. [10] phase, the best separation of components was observed,
showing 5 spots.
The study aims to investigate the phytochemical and
antimicrobial properties of the extracts of the leaf of the  Fractionation
Nigerian Polyalthia longifolia used in ethnomedicine. Aqueous methanol (50% H20 + 50% MeOH) was adde
d into a 250 mL pre-weighed beaker containing the crude
II. MATERIALS AND METHODS methanolic extract. After a few minutes of standing, the
mixture was then extracted with hexane (2x 200 cm3) using a
 Plant Material Collection and Identification separatory funnel to obtain the hexane fraction. 70 mL of
Polyalthia longifolia leaves were harvested in March CHCl2 was added to the Aq. MeOH layer, and then extracted
2025 from Veritas University, Abuja. The leaves were with 1 M HCl (2x100 cm3) to obtain CHCl2 layer and Aq.
authenticated at the National Institute for Pharmaceutical Acid-soluble layer. The CHCl2 layer was also extracted with
Research and Development (NIPRD), Idu and a specimen 1 M NaHCO3 (2x100cm3) to obtain the CHCl2 layer fraction
was deposited with voucher number NIPRD/H/7474. The (neutrals) and the Aq. Layer, which was acidified with 1 M
leaves were weighed and washed with running distilled water. HCl, and then extracted with chloroform (2x 200 cm 3) to
They were dried on a lab bench for two weeks at room obtain the acid fraction. The Aq. Acid soluble layer was
temperature and without sunlight. Then, using an electric basified with NH3(aq) and then extracted with chloroform
blender, the dried plant material was blended into powder. (2x250 cm3) to obtain the base fraction (Figure 1). Each
fraction was weighed after evaporating to dryness. TLC of the
 Extraction of Dried Powdered Plant Aq. MeOH layer was done using an aluminum sheet pre-
In a glass container, the dried powdered leaf of coated with silica gel and 1:5 ethyl acetate/hexane solvent
Polyalthia longifolia was cold macerated for 48 hours with system gave the best separation and showed 6 number of
intermittent shaking using 42.0 L of methanol. The extract spots.

Fig 1 Scheme for Fractionation of the Crude Methanolic Extract of Polyalthia longifolia

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Volume 10, Issue 7, July – 2025 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165 https://doi.org/10.38124/ijisrt/25jul1959
 Preliminary Phytochemical Screening determined by taking the least concentration in the series that
A preliminary qualitative phytochemical screening was showed no growth or apparent sign.
performed on Polyalthia longifolia's crude methanolic
extract. Using standard procedure [11], the crude extract was  Minimum Bacteriocidal Concentration (MBC)
tested to identify eight secondary metabolites, including Determination
tannins, alkaloids, carbohydrates, saponins, sterols/steroids, The findings from the MIC test were utilized to
flavonoids, volatile oils, and terpenoids. ascertain the MBC of the extract The test tubes that did not
exhibit any turbidity (clear) in the MIC test were filled with a
 Antimicrobial Screening sterilised wire loop, and a loopful was streaked over sterile
The antimicrobial screenings, including sensitivity tests, nutrient agar plates. For 18 to 24 hours, the plates were
minimum inhibitory concentration (MIC) and minimum incubated at 37 °C. The plates were examined for the
bacteriocidal concentration (MBC) were performed using a presence or absence of growth following the incubation time.
standard procedure[11]. This evaluation aimed to determine if the extract's
antibacterial activity was bacteriostatic or bacteriocidal
 Sensitivity Test for Determining Inhibitory Activity
The sensitivity test was done by the agar well diffusion  FT-IR and GC-MS Analyses of Crude Methanolic Extract
method . The standardised inocula of the bacterial and fungal of P. longifolia
isolates were streaked onto sterilised Mueller Hinton and FT-IR and GC-MS analyses were carried out on the
Potatoe dextrose agar plates, respectively, with a sterile swab crude methanolic extract of Polyalthia longifolia using a
stick. Using a sterile cork borer, four wells were punched into Nicolet iS10 FTIR Spectrophotometer equipped with Win-IR
each inoculated agar plate. The wells were properly labelled Pro Version software and a Perkin Elmer Turbo mass
based on the concentrations of the extract prepared (100 spectrometer (Norwalk, CTO6859, USA), respectively.
mg/ml, 50 mg/ml, 25 mg/ml, and 12.5 mg/ml, respectively).
The extract (approximately 0.2 mL) was added to each well. III. RESULTS AND DISCUSSION

For about an hour, the inoculated plates with the extract  Extraction and Fractionation
were placed on the bench to allow the extract to permeate the A dark green coloration was obtained from crude extract
agar. with the highest yield (26.47 g). The result of cold methanol
maceration from 520.83 g of dried leaves, produced a yield
Incubation of the Muller Hinton agar plates was done of about 5.08% relative to the plant material. Fractionation of
for 24 hours at 37°C. At room temperature, the potato the crude methanolic extract yielded hexane-soluble, acid,
dextrose agar plates were incubated for about 3 to 5 days. base, and neutral fractions in varying small amounts. The
base fraction had the highest yield among the fractions (0.539
After that, the incubated plates were checked for any g, 2.036% of the crude), suggesting a higher presence of basic
signs of inhibition, which showed up as a clear zone of compounds, such as alkaloids. The acid and hexane fractions
inhibition encircling the wells. had nearly equal yields (~1%), while the neutral fraction
yielded the least (0.233 g, 0.880%) (Table 1).
A clear ruler that was calibrated in millimetres was use
d to measure these inhibitory zones' diameter. Most fractions were gummous in nature, though their
color and shine differed: The hexane and neutral fractions
 Minimum Inhibitory Concentration (MIC) Determination resembled the crude extract in color (dark green). The acid
Mueller Hinton Broth was employed as a diluent in the and base fractions were yellowish with a shiny texture,
tube dilution method to determine the extracts' minimum indicating likely differences in compound composition and
inhibitory concentration. polarity. The fractionation procedure was successful in
separating components based on polarity. The relatively high
In a test tube filled with Mueller Hinton broth, the yield of the base fraction correlates with the phytochemical
extract was serially diluted to the lowest dose that inhibited results showing alkaloid presence and with antimicrobial
each organism during the sensitivity test. In each tube results where the base fraction showed strong activity. These
containing the broth and extract, the standardised organisms differences in yield and appearance hint at varying
were added. For twenty-four hours, the inoculated tubes were concentrations and types of phytochemicals, which were later
incubated at 37 °C. Using turbidity as a criteria, the tubes validated by antimicrobial screening and chemical analyses
were inspected for growth at the conclusion of the incubation (GC-MS).
time. The minimum inhibitory concentration (MIC) was

Table 1 Physical Features and Yields of Extracs from Polyalthia longifolia Leaves
Extractives Yield (g) and % Relative to Crude Colour and Consistency
Crude 26.47 (5.082) Dark green gum
Hexane-soluble fraction 0.262 (0.989) Dark green gum
Acid fraction 0.269 (1.016) Light yellow shiny gum
Base fraction 0.539 (2.036) Yellow shiny gum
Neutrals 0.233 (0.880) Dark green gum

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Volume 10, Issue 7, July – 2025 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165 https://doi.org/10.38124/ijisrt/25jul1959
 Phytochemical Analysis disrupting cell membranes and inhibiting microbial enzymes
The phytochemical analysis of the leaf showed that [15]. Terpenoids inhibit microbial growth by affecting
tannins, alkaloids, saponins, volatile oils, terpenoids, membrane integrity and metabolic processes [16]. Flavonoids
flavonoids, and steroids were present (Table 2). These contribute to antimicrobial and antioxidant activities by
phytochemicals are known for their broad spectrum of interfering with nucleic acid synthesis and cell membrane
biological activities. Tannins possess antimicrobial and function [17]. Furthermore, steroids have been demonstrated
antioxidant properties by forming complexes with microbial to possess anti-inflammatory properties and can influence the
proteins and enzymes [12]. Alkaloids exhibit antimicrobial structural integrity of microbial cells [18]. The presence of
action by disrupting DNA replication and microbial these compounds in P. longifolia supports its traditional use
metabolism [13]. Saponins damage microbial membranes by in herbal medicine and provides a scientific basis for its
interacting with sterols, causing increased permeability [14]. observed antimicrobial activity.
Volatile oils, or essential oils, exert antimicrobial effects by

Table 2 Phytochemical Analysis of the Crude Extract of Polyalthia longifolia Leaf

Metabolites Results
Tannins +
Alkaloids +
Saponins +
Volatile oils +
Terpenoids +
Flavonoids +
Steroids +
Key: (+) = Present

 Antimicrobial Screening of Crude Extract and Fractions suggest bactericidal effects. Conversely, the acid fraction
Antimicrobial screening of the showed high MIC and MBC values, or no measurable effect
crude extract and fractions of Polyalthia longifolia at all, indicating weak antibacterial potential (Table 4).
demonstrated potent antibacterial properties against bacteria
of both gram-positive and gram-negative The neutral fraction exhibited particularly strong
types, especially in the crude and neutral fractions.(Table 3). antibacterial effects, with inhibition zones reaching up to 33
These fractions exhibited a concentration dependent mm against Staphylococcus aureus, surpassing results from
inhibitory effect, with larger zones of inhibition observed at earlier studies involving methanolic or aqueous extracts [19].
higher concentrations. The strongest antibacterial activity This suggests that the neutral, pH-balanced fraction may
was found in the neutral fraction, with a zone of inhibition of concentrate or preserve potent antibacterial compounds, a
33 mm against Staphylococcus aureus at 100 mg/ml, closely finding that has been rarely documented and merits further
comparable to the 37 mm zone produced by the standard investigation.
antibiotic, Ciprofloxacin. Similarly, the neutral fraction was
highly effective against Salmonella typhi, Bacillus subtilis, Of significance, is the inactivity of the crude extract and
and Escherichia coli, having inhibition zones of 30 mm, 32 fractions towards the fungi Candida albicans and Aspergillus
mm, and 28 mm, respectively (Table 3). The crude fraction niger. While earlier studies have reported antifungal effects
also demonstrated strong antibacterial effects across the same of P. longifolia [20, 10], this inconsistent finding may be due
organisms, though slightly lower than the neutral fraction. to variation in soil composition and environmental
The hexane and base fractions displayed moderate circumstances. Furthermore, the base fraction demonstrated a
antibacterial activity. Notably, the base fraction produced a notably high zone of inhibition (33 mm) against Salmonella
high inhibition zone of 33 mm against Salmonella typhi, typhi. This may point to the presence of basic or non-polar
suggesting strong bactericidal action. In contrast, acid compounds particularly effective against S. typhi.
fraction exhibited minimal antibacterial activity, with little or Additionally, MIC and MBC values for the neutral and crude
no zones of inhibition, especially at lower concentrations. fractions were low, with MICs as low as 3.125 mg/ml,
indicating potent bactericidal properties. These values are
MIC and MBC results confirmed the antibacterial equal to or lower than previously reported figures [21],
potential of the crude and neutral fractions, with low MBC suggesting that the methods used in this study may yield more
values between 6.25 and 12.5 mg/ml and MIC values between concentrated or bioavailable forms of active constituents.
3.125 and 6.25 mg/ml.. These close MIC and MBC values

Table 3 Inhibitory Activity of the Crude Extract and Fractions of P. longifolia

Zone of inhibition (mm) at different extract concentrations (mg/ml)
Test organisms crude neutrals hexane base acid C
100 50 25 12.5 100 50 25 12.5 100 50 25 12.5 100 50 25 12.5 100 50 25 12.5
27 25 21 19 33 29 27 24 30 25 23 21 23 20 18 16 18 15 12 - 37
S. aureus
29 27 25 22 32 27 24 21 27 23 20 18 21 19 17 14 16 14 10 - 30
B. subtillis
26 23 21 18 28 26 23 20 22 20 17 15 22 19 16 15 - - - - 33
E. coli

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28 25 22 20 30 27 24 22 28 26 22 22 33 29 26 22 - - - - 35
S. typhi
- - - - - - - - - - - - - - - - - - - - 46
A. niger
- - - - - - - - - - - - - - - - - - - - - 38
C. albicans
Key: (-) = No activity
C = Ciprofloxacin/Econazole (Control)

Table 4 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration

(MBC) / Fungicidal Concentration (M.F.C) of the extracts
TEST MIC MBC
ORGANISMS
Crude Neutrals Hexane Base Acid Crude Neutral Hexane Base Acid
3.12 3.125 3.125 12.5 25 6.25 6.25 6.25 25 50
S. aureus
6.25 12.5 6.25 6.25 50 12.5 25 12.5 12.5 NIL
B. substilis
3.125 6.25 12.5 25 _ 6.25 12.5 25 50 _
E. coli
3.125 6.25 12.5 6.25 _ 6.125 12.5 25 12.5 _
S. typhi
_ _ _ _ _ _ _ _
C. albicans - -
_ _ _ _ _ _ _ _
A .niger - -
Key: (-) = Not determined for M.I.C/M.B.C
NIL= No M.B.C ( The extract is bacteriostatic not bactericidal)

 FT-IR and GC-MS Analyses of Crude Methanolic Extract flavonoids, confirming the results of the phytochemical
The FT-IR spectrum of the crude methanolic extract of screening that showed phenolic compounds and flavonoids
Polyalthia longifolia (Figure 2) provided valuable insight into were present. The absorptions band at 2825.3 cm-1 shows
the functional groups present in the plant extract. aliphatic C–H stretching vibrations, which suggests the
presence of methyl (-C-H) groups while the band at 2918.5
Around 3300–3400 cm⁻¹, a considerable absorption cm-1 corresponds to that of the methylene groups (=C-H)
band is observed, which suggests the existence of hydroxyl typically found in phenols, flavonoids, and terpenoids.
groups (–OH), which are common in alcohols, phenols, and

Fig 2 FT-IR Spectrum for Crude Methanolic Extract of P. longifolia

A sharp peak observed near 1700–1750 cm⁻¹ indicates The gas chromatogram of the crude extract P. longifolia
the presence of carbonyl (C=O) functional groups, consistent revealed sixteen peaks based on a 1% area percentage and a
with the fatty acids such as oleic and palmitic acids identified 27% quality of characterisation (Figure 3). The sample's mass
in the GC-MS analysis. In the region around 1600–1650 cm⁻¹, spectra were utilised to identify the structure and nature of the
peaks associated with C=C stretching vibrations point to the compounds by comparing them to standards from the
presence of aromatic compounds like flavonoids and other database (Figure 4). The major compounds identified include:
benzene derivatives. mesitylene, benzene,1,2,4-trimethyl, n-hexadecanoic acid,
dibutyl phthalate, 1-Cyclohexene-1- acetaldehyde,2,6,6-
The presence of strong and sharp band at 1013.8 cm -1 trimethyl, 1,3,6,10-Cyclotetra-decatetraene,3,7,11-trimethyl-
indicates the presence of the C-O functional group present in 14-(1-methylethyl)-,[S-(E,Z,E,E)]-, oleic acid, octadecanoic
majority of the compounds identified in the physicochemical acid, methyl kolavenate, farnesol formate, diisooctyl
characterization. phthalate, benzamide, 2-fluoro-N-(2,4-dimethoxyphenyl),

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9,19-cyclolanostan-3-ol,acetate, (3.beta.), 5-ethylcyclopent- dimethoxyphenyl), implies the presence of phenolic
1-ene-1-carboxylic acid, naphthalene, decahydro-, 1,6- derivatives often seen in flavonoid structures [4]. Volatile
bis(methylene)-4-(1-methylethyl)-,(4.alpha., 4a.alpha.,8a. constituents such as mesitylene, 1-cyclohexene acetaldehyde,
alpha.)- and 26-nor-5-cholesten- 3.beta.-ol-25-one (Table 5). and naphthalene derivatives account for the volatile oils
detected in the phytochemical test. These compounds possess
Compounds such as farnesol formate, 1,3,6,10- antimicrobial properties and are also associated with
cyclotetradecatetraene, and methyl kolavenate are typical oxidative stress protection [5]. The nitrogen-containing
terpenoid structures known for their anti-inflammatory and compound (benzamide derivative) hints at alkaloid-like
antimicrobial activities [4]. Additionally, complex structures, reinforcing the ethnopharmacological relevance of
triterpenoids such as 26-nor-5-cholesten-3β-ol-25-one and the plant. Tannins, known for their astringent and
9,19-cyclolanostan-3-ol acetate represent steroidal antimicrobial effects, may not have appeared in the
backbones and confirm the presence of phytosterols, which chromatogram due to their non-volatile nature but remain
have been associated with cholesterol-lowering and cytotoxic relevant based on the test results.
effects [5]. The identification of aromatic hydrocarbons and
substituted benzene derivatives such as mesitylene and Furthermore, fatty acids such as oleic acid, n-
benzene-1,2,4-trimethyl suggests the presence of phenolic hexadecanoic acid and octadecanoic acid were identified in
compounds, consistent with the phytochemical detection of high percentages. These fatty acids are known for their anti-
flavonoids and tannins. While flavonoids were not directly inflammatory, antioxidant, and membrane-protective effects
named in the GC-MS output, the presence of methoxy- [22].
substituted benzamides, like benzamide, 2-fluoro-N-(2,4-

Fig 3 Gas Chromatogram for Crude Methanolic Extract of P. longifolia

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ISSN No:-2456-2165 https://doi.org/10.38124/ijisrt/25jul1959

n-Hexadecanoic Acid Dibutyl Phthalate

Methyl Kolavenate Farnesol Formate

Diisooctyl Phthalate 9,19-Cyclolanostan- 3-ol, Acetate, (3.beta.)

Fig 4 MS Spectra and Structures of some Components of the Crude Methanolic Extract of P.longifolia

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Table 5 The GC-MS Analysis of the Crude Methanolic Extract of P. longifolia, Showing its Major Compounds

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IV. CONCLUSION Research, 246, 118061.
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In conclusion, these findings demonstrate the potential [11]. Obi, L. K., & Okwute, S. K. (2023). Chemical and
of P. longifolia as a natural source of antibacterial compounds Biological Investigations of the Leaf Extracts of
and offer scientific support for its traditional usage in herbal Andrographis paniculata (Acanthaceae). Journal
medicine. However, the extracts showed no activity against ISSN, 2766, 2276.
Aspergillus niger and Candida albicans, indicating a narrower [12]. Scalbert, A. (1991). Antimicrobial properties of
spectrum of antifungal potential. tannins. Phytochemistry, 30(12), 3875–3883.
[13]. Cowan, M.M. (1999). Plant products as antimicrobial
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