Hindawi

Evidence-Based Complementary and Alternative Medicine

Volume 2017, Article ID 1517683, 10 pages
[Link]

Research Article
GC-MS Based Metabolite Profiling, Antioxidant and
Antimicrobial Properties of Different Solvent Extracts of
Malaysian Plectranthus amboinicus Leaves

Mallappa Kumara Swamy,1 Greetha Arumugam,1 Ravinder Kaur,1 Ali Ghasemzadeh,1

Mazina Mohd. Yusoff,2 and Uma Rani Sinniah1
1
Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
2
Laboratory of Natural Products, Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

Correspondence should be addressed to Mallappa Kumara Swamy; [Link]@[Link]

and Uma Rani Sinniah; umarani@[Link]

Received 8 December 2016; Accepted 9 March 2017; Published 23 March 2017

Academic Editor: Dolores Garcı́a Giménez

Copyright © 2017 Mallappa Kumara Swamy et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

This study evaluates the phytochemistry, antioxidant, and antimicrobial effects of Plectranthus amboinicus leaves extracted in
different solvents. The methanol extract contained the highest total phenolic (94.37 ± 1.24 mg GAE/g) and flavonoid contents
(26.90 ± 1.35 mg RE/g) and exhibited the highest DPPH scavenging activity (90.13 ± 3.32%) followed by the acetone extract (80.23 ±
3.26%) at 500 𝜇g/mL concentration. Similarly, the highest ferric ion reduction potential (849.63 ± 30.95 𝜇M of Fe (II)/g dry weight)
was exhibited by the methanol extract followed by the acetone extract (695.92 ± 25.44 𝜇M of Fe (II)/g dry weight). The methanol
extract showed greater antimicrobial activity against all the tested pathogens (Bacillus subtilis, Methicillin-resistant Staphylococcus
aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans). However, both hexane and acetone extracts failed to
inhibit E. coli. S. aureus and C. albicans were more susceptible to all the extracts. Further, GC-MS analysis confirmed the occurrence
of a total 46 phytocompounds in different solvent extracts. Some of the major compounds included carvacrol (37.7%), tetracontane
(16.6%), squalene (15.6%), tetrapentacontane (13.7%), and Phytol (12.9%). In conclusion, extraction solvents influenced the recovery
of phytocompounds and the highest pharmacological activities of the methanol extract could be correlated to the presence of
additional bioactive compounds.

1. Introduction However, synthetic antioxidants cause adverse effects on the

human body. Currently, though natural drugs are developed
In nature, plants encompass a wide range of therapeutically against human pathogens, the occurrence of drug resistance
valued bioactive compounds. These natural compounds are in microbes is posing a major challenge to the scientific
widely used in both traditional and modern therapies for community. Therefore, there is a need to discover novel drugs
improving human health with relatively less or no side effects. from the natural sources to benefit mankind. Plectranthus
Globally, various medicinal plants have been well explored to amboinicus (Lour.) Spreng. is a medicinal herb (Lamiaceae)
discover novel drug molecules to combat the threat of ever- which grows naturally in the subtropics and tropics of Africa,
increasing human diseases [1–3]. In the human body, cellular Asia, and Australia. The occurrence of about 30 nonvolatile
mechanisms lead to the generation of unstable compounds and 76 volatile compounds attributes to its pharmacological
such as reactive oxygen species and free radicals, which can properties such as anti-inflammatory, antimicrobial, wound
destroy living cells and may cause several clinical diseases. healing, analgesic, antitumor, antiepileptic, antioxidant, and
A range of synthetic and natural antioxidants are proved to larvicidal actions [2]. Recent studies have suggested the suc-
be effective in controlling the activities of free radicals [4]. cess of this herb against oral, respiratory, skin, cardiovascular,
2 Evidence-Based Complementary and Alternative Medicine

digestive, and urinary diseases [2]. Previous studies have content and expressed in mg GAE (gallic acid equivalents)
witnessed the existence of significant variations in secondary per gram of dried solvent extract.
metabolites accumulation among the same plant species
grown under diverse ecological conditions [5, 6]. In general, 2.4. Determination of Total Flavonoid Content in P. amboinicus
various solvents are used for extracting plant metabolites Extracts. The presence of total flavonoids content in various
from their different parts. Moreover, the extraction of phy- solvent extracts of P. amboinicus leaves was determined by
tocompounds and yield mainly depends on the type of using the colorimeter assay method as described by Swamy et
solvents and the method of extraction [7]. Literature survey al. [4]. In short, about 0.5 mL of the solvent extract (1 mg/mL)
shows less information on phytochemicals and bioactivities was suspended in 2 mL of distilled water and then added
of P. amboinicus plants growing under tropical conditions of with 150 𝜇L of 5% NaNO2 solution and incubated at room
Malaysia [8]. With this background, we investigated the influ- temperature for 5 min. Thereafter, the solution was added to
ence of different solvents to recover higher phytochemicals 2 mL of NaOH (4%) and 600 𝜇L of AlCl3 (10%) and made up
from a local P. amboinicus plant leaves and assessed their to 5 mL using distilled water. The solution was mixed thor-
polyphenols and antioxidant and antimicrobial activities. oughly and incubated for 15 min at room temperature. The
Further, GC-MS analysis was carried out to explain the absorbance of the solution was documented at a wavelength
occurrence of bioactive nonvolatile compounds in different of 510 nm against the blank (water). In a similar way, a
solvent extracts. standard calibration curve was prepared by using the stan-
dard, rutin, at different concentrations. By using the standard
2. Materials and Methods curve of rutin, total flavonoid content was determined and
expressed in mg of RE (rutin equivalents) per gram of dried
2.1. Plant Material Collection. The wild growing P. amboini- solvent extract.
cus plant material was collected from the forest area near Uni-
versiti Putra Malaysia (UPM), Serdang, Selangor, Malaysia, 2.5. In Vitro Antioxidant Activity Evaluation of
during the month of August 2015. The plant was identified
P. amboinicus Extracts
and authenticated taxonomically at the Department of Crop
Science, UPM, Malaysia. The voucher specimen, PA-082015, 2.5.1. DPPH Free Radical Scavenging Assay. 1,1-Diphenyl-2-
was deposited in the department. The detached leaves from picrylhydrazyl (DPPH) free radical scavenging assay was
the collected plant materials were washed carefully under used to evaluate the antioxidant activity of different solvent
running tap water and dried for 1 week at room temperature extracts of P. amboinicus leaves as explained by Mohanty et al.
in a shaded area. The dried leaves were finely powdered by [9] with minor modifications. In brief, different plant extract
using an electric blender and stored in a plastic bag container in the same volume (0.3 mL) at different concentrations
for further use at room temperature. ranging from 100 to 1000 𝜇g/mL was added to 2 mL of DPPH
solution (0.1 mM) and placed in dark condition at room tem-
2.2. Preparation of Extracts. About 25 grams of powdered perature for 30 min. The absorbance of this solution was mea-
leaves of P. amboinicus were kept in a beaker to which 100 mL sured at a wavelength of 517 nm against the blank (methanol)
of various organic solvents (methanol, acetone, and hexane) by using UV-visible spectrophotometer. Ascorbic acid was
was added and thoroughly shaken. Later, the mixture was used as the standard control. The percentage of inhibiting free
placed at room temperature for 48 hrs and stirred 2-3 times a radicals by each extracts was calculated by using the equation
day. After filtering the mixture, the filtrate was evaporated to given below:
dryness using rota-vapor. The final extracts were weighed to
determine the yield (%) and the dried extracts were stored at Inhibition activity (%)
4∘ C in a refrigerator for further studies. Absorbance (control) − Absorbance (extract)
= (1)
Absorbance (control)
2.3. Determination of Total Phenolic Content in P. amboinicus
Extracts. Using the FC (Folin-Ciocalteu) colorimetric assay × 100.
method, total phenolic content occurring in different solvent
extracts of P. amboinicus leaves was evaluated [4]. Briefly, 2.5.2. FRAP (Ferric Reducing Antioxidant Potential) Assay.
dried plant extract (0.1 g) was dissolved in 1 mL of distilled FRAP assay was used to determine the total antioxidant activ-
water. About 0.1 mL of this solution was later added to a solu- ity of different solvent extracts of P. amboinicus leaves. Briefly,
tion containing 20% sodium carbonate solution (1 mL) and FRAP reagent was freshly prepared by mixing 2.5 mL of FeCl3
50% FC reagent (0.5 mL). This solution was mixed thoroughly (20 mmol/L), 2.5 mL of 10 mmol/L TPTZ (2,4,6-tripyridyl-
and allowed to stand by at room temperature for 20 min to S-triazine), and 25 mL of 0.3 mol/L acetate buffer (pH, 3.6)
facilitate the reaction to occur. Afterwards, the absorbance of and incubated at 37∘ C under dark condition for 20 min.
the solution was recorded at a wavelength of 730 nm against Different solvent extracts (2.0 mL) were added into 2.0 mL of
the blank (water). Likewise, a standard calibration curve FRAP reagent and the final volume was made up to 10 mL.
was generated by using gallic acid (standard) at different The mixture was incubated at 25∘ C for 30 min under dark
concentrations. The gallic acid curve was used to convert condition. The absorbance of the solution was recorded at
the absorbance of different plant extracts into total phenolic 593 nm using acetate buffer as the blank. Results are expressed
Evidence-Based Complementary and Alternative Medicine 3

in 𝜇M Fe (II)/g dry weight and equated with the standard, 𝛼- Table 1: Dry weight and total yield of different solvent extracts of P.
tocopherol. amboinicus leaves.
Solvent extracts Weight of the extract∗ (mg ± SD) Yield (%)
2.6. Determination of Antibacterial Activity. The test included Methanol 351.53 ± 4.31a 4.0
a total of four bacterial species, namely, Bacillus subtilis Acetone 111.6 ± 3.01c 1.0
B29, Methicillin-resistant Staphylococcus aureus (MRSA) Hexane 131.56 ± 2.69b 1.32
(ATCC700698) (gram-positive), Pseudomonas aeruginosa ∗
Each value is expressed as mean ± standard deviation (SD) (𝑛 = 3).
(ATCC 15442), Escherichia coli E266 (gram-negative), and Values in the column followed by a different letter superscript are signifi-
one fungal species, Candida albicans 90028. All microbial cantly different (𝑝 < 0.05).
strains were procured from the Laboratory of Molecular
Biomedicine, Institute of Bioscience, UPM, Serdang, Malay- Table 2: Total phenolics and flavonoids content of different solvent
sia. The pure cultures of bacterial strains were subcultured extracts of P. amboinicus leaves.
onto Müller-Hinton Agar (MHA) while C. albicans 9002 was
cultured on potato dextrose agar (PDA). The antibacterial Total phenolic Total flavonoid
properties of different solvent extracts of P. amboinicus leaves Solvent extracts content∗ content∗
were evaluated by using disc diffusion method as described (mg GAE/g) ± SD (mg RE/g) ± SD
by Kumara et al. [10] with little modifications. In short, 10 mg Methanol 94.37 ± 1.24a 26.90 ± 1.35a
of each solvent extract was dissolved in 1 mL of dimethyl Acetone 63.20 ± 1.22c 21.27 ± 0.80b
sulfoxide (DMSO) and about 10, 20, and 30 𝜇L of this solution Hexane 75.39 ± 1.07b 16.46 ± 0.99c
were impregnated on 6 mm sized sterilized filter paper discs. ∗
Each value is expressed as mean ± standard deviation (SD) (𝑛 = 3). Values
Later, the air-dried discs were placed on individual MHA in the column followed by a different letter superscript are significantly
or PDA plates and previously preinoculated uniformly with different (𝑝 < 0.05) and values having the same letters are not statistically
a known bacterial or fungal strain, respectively. The discs significant (𝑝 < 0.05). GAE: gallic acid equivalent and RE: rutin equivalent.
saturated with DMSO (20 𝜇L) served as a negative control for
all microbes while streptomycin (100 mg/mL) and nystatin
(100 mg/mL) served as a positive control for bacterial and (Table 1). The methanol solvent recorded the highest weight
fungal species, respectively. All microbes were incubated in of the extract (131.6 ± 2.6 mg) and its yield (1.3%). The
an incubator at 37∘ C for 24 hrs to observe the zone of growth extractable elements recovered from different solvents are in
inhibition (mm) around each disc. For each microbial strain, the following order, that is, methanol > hexane > acetone.
the experiment was repeated 3 times. Further, solvent extracts had a significant difference (𝑝 <
0.05) in their total phenolic and total flavonoid content
(Table 2). In the methanol leaf extract, the highest total
2.7. GC-MS Analysis. Each solvent extract was subjected to
phenolic content (94.37 ± 1.24 mg GAE/g) and flavonoid
GC-MS analysis using the model instrument, GCMS-QP2010
content (26.9±1.3 mg RE/g) were recorded. In hexane extract,
Ultra (Shimadzu Co., Japan) attached with a capillary column
total phenolic content was found to be 75.39±1.07 mg GAE/g.
DB-1 (0.25 𝜇m film × 0.25 mm I. d. × 30 m length). Analysis
However, total flavonoid content (16.46 ± 0.9 mg RE/g)
was performed by injecting 1 𝜇L of the sample with a split
was relatively lesser when compared to the acetone extract
ratio of 20 : 1. Helium gas (99.9%) was used as the carrier gas at
(21.27 ± 0.8 mg RE/g).
a flow rate of 1 mL/min. The analysis was performed in the EI
DPPH free radical scavenging property was observed to
(electron impact) mode with 70 eV of ionization energy. The
be dependent on the concentration for all the tested solvent
injector temperature was maintained at 250∘ C (constant). The
extracts. The methanol extract exhibited the highest DPPH
column oven temperature was set at 50∘ C (held for 3 min),
scavenging activity (90.13 ± 3.32%) followed by acetone
raised at 10∘ C per min to 280∘ C (held for 3 min), and finally
extract (80.23 ± 3.26%) and hexane extract (73.52 ± 3.18%)
held at 300∘ C for 10 min. The compounds were identified after
at 500 𝜇g/mL concentration (Figure 1).
comparing the spectral configurations obtained with that of
However, DPPH scavenging activity of all extracts was
available mass spectral database (NIST and WILEY libraries).
inferior to the standard (ascorbic acid). Similar pattern of
antioxidant activity was evidenced from FRAP assay method
2.8. Statistical Analysis. In each experiment, the data where the highest ferric ion reduction potential was exhibited
recorded was from 3 replications (𝑛 = 3) and all the results by the methanol extract (849.63 ± 30.95 𝜇M of Fe (II)/g dry
are represented as mean ± SD. One-way analysis of variance weight) followed by acetone extract with 695.92±25.44 𝜇M of
(ANOVA) was carried out to compare the data. Further, to Fe (II)/g dry weight (Figure 2). The lowest FRAP activity was
determine the statistically significant differences, Tukey’s observed in hexane extract (376.98±15.42 𝜇M of Fe (II)/g dry
test was performed at 𝑝 < 0.05 level using GraphPad Prism weight). However, the superior activity was evidenced in the
(version 5.0) statistical software. standard, 𝛼-tocopherol.
The microbial inhibitory potential of different solvent
3. Results extracts of P. amboinicus is depicted in Table 3. The results
clearly showed the existence of a varied and selective antimi-
The dry weight and final yield of the leaf extract were signif- crobial activity of different solvent extracts against each
icantly affected by different solvents used for the extraction microbial species tested. Further, increased concentration
4 Evidence-Based Complementary and Alternative Medicine

Table 3: Antibacterial activity of different solvent extracts of P. amboinicus at different concentrations.

Zone of inhibition∗ (mm)

Solvent extracts Pseudomonas
(𝜇g/disc) Bacillus subtilis Staphylococcus aureus Escherichia coli Candida albicans
∗∗ aeruginosa
B29 (𝑀𝑅𝑆𝐴) E266 90028
ATCC 15442
Hexane
100 06.1 ± 0.1 08.0 ± 0.3 03.9 ± 0.3 — 06.1 ± 0.2
200 06.9 ± 0.4 09.2 ± 0.2 06.0 ± 0.2 — 06.9 ± 0.4
300 08.2 ± 0.1 10.0 ± 0.3 07.0 ± 0.2 — 07.9 ± 0.3
Methanol
100 08.1 ± 0.2 07.1 ± 0.5 06.1 ± 0.2 06.0 ± 0.2 07.1 ± 0.2
200 08.9 ± 0.1 08.0 ± 0.5 06.2 ± 0.3 07.4 ± 0.5 08.3 ± 0.3
300 10.2 ± 0.5 09.2 ± 0.3 07.2 ± 0.2 08.7 ± 0.6 09.0 ± 0.3
Acetone
100 01.9 ± 0.1 05.2 ± 0.3 02.2 ± 0.2 — 05.6 ± 0.3
200 02.9 ± 0.2 06.1 ± 0.4 03.2 ± 0.3 — 06.0 ± 0.2
300 03.0 ± 0.1 08.4 ± 0.5 03.9 ± 0.2 — 06.8 ± 0.3
∗
The experiment included DMSO (20 𝜇L) as negative control while streptomycin (100 mg/mL) for bacteria and nystatin (100 mg/mL) for yeast served as positive
control. Each value represents the mean ± standard deviation (SD) of 3 replicates per treatment in 3 repeated experiments.
“—” represents no activity observed.
∗∗
MR represents Methicillin resistant.

120 1200
(mol Fe (II)/g dry weight)

100 1000
FRAP activity
Inhibition (%)

80 800

60 600

40 400
200
20
0
0 50 100 200 400 800
50 100 200 400 800
Concentration (g/mL)
Concentration (g/mL)
Methanol extract Hexane extract
Methanol extract Hexane extract
Acetone extract Tocopherol
Acetone extract Tocopherol
Figure 2: FRAP assay of various solvent extracts of P. amboinicus.
Figure 1: DPPH free radical scavenging activities of various solvent
extracts of P. amboinicus.

belong to different chemical classes and most of them are

of extracts evidenced relatively a higher activity in all the reported to exhibit important biological activities. Figure 3
tested microbes irrespective of the solvent. Among all the shows the distinct chromatogram of P. amboinicus leaves
extracted in different solvents. The identified compounds
tested microbes, S. aureus (MRSA) and C. albicans were
with their peak number, retention time (RT), and peak area
more susceptible to all the extracts evaluated. The highest
(%) are presented in Table 4. Out of 39 peaks observed
activity was observed in the methanol extract for B. subtilis in the chromatogram of methanol extract, 19 compounds
(10.2 ± 0.5 mm), S. aureus (MRSA) (09.2 ± 0.3 mm), P. were identified with 6 major peaks. The major compounds
aeruginosa ATCC 15442 (07.2 ± 0.2 mm), E. coli E266 (08.7 ± included tetracontane (16.6%), tetrapentacontane (11.3%),
0.6 mm), and C. albicans (09.0 ± 0.3 mm) at a concentration pentacosane (7.8%), and n-hexadecanoic acid (4.9%).
of 300 𝜇g/disc. However, both hexane and acetone extracts Among the 26 peaks observed in the GC-MS profile of
failed to inhibit E. coli E266. acetone extract, only 11 compounds were detected. The major
Further, GC-MS profiling of the extracts together compounds identified were Phytol (12.9%), squalene (15.6%),
revealed the occurrence of a total of 115 peaks of which about and 𝛽-Amyrin (5.3%). While hexane extract profile revealed
46 chemical compounds were identified. These compounds the presence of 16 detectable compounds from a total of
Evidence-Based Complementary and Alternative Medicine 5

TIC

31

36
34
24

38
8

27
10

33
13

37
35
32

39
5

28
26 25
212022

29
30
15
16

23
14
6

17
1

19
7

18
11 12
3

9
4
2

10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0

(min)
(a)

14

26
24
1

22

25
18 19 17
11

21
23
20
10 9

16
13

15
78 6
5

12
3
2

20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0

(min)
(b)
TIC
4

20
65

14
10
9

17
1615
18
7

1213
11

19
32

8
1

10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0

(min)
(c)

Figure 3: GC-MS based chemical profiling of methanol (a), acetone (b), and hexane (c) extracts from P. amboinicus leaves.
6 Evidence-Based Complementary and Alternative Medicine

Table 4: GC-MS profile of different solvent extracts of P. amboinicus leaves.

S. number Name of the compound Peak number R. time Peak area (%)
Methanol extract
(1) Butanoic acid, methyl ester 2 03.14 00.35
(2) 5-Methoxypyrrolidin-2-one 3 19.69 00.72
(3) (−)-Loliolide 5 45.52 02.15
(4) Neophytadiene 7 46.75 00.81
(5) 2-Pentadecanone, 6,10,14-trimethyl- 8 47.11 03.01
(6) 1-Decanol, 2-hexyl- 9 49.84 00.49
(7) n-Hexadecanoic acid 10 51.46 04.97
(8) Methyl (Z)-5,11,14,17-eicosatetraenoate 12 55.93 00.50
(9) Phytol 13 56.13 03.30
(10) Phytol, acetate 15 59.30 00.61
(11) Octanoic acid, 2-dimethylaminoethyl ester 17 61.48 00.39
(12) Carbonic acid, 2-dimethylaminoethyl isobutyl ester 19 66.60 00.51
(13) Di-n-octyl phthalate 21 68.59 00.66
(14) Tetrapentacontane 24 72.63 11.32
(15) Pentacosane 27 75.78 07.88
(16) Triacontane 28 80.93 02.60
(17) Tetracontane 31 83.42 16.67
(18) Squalane 35 86.54 01.73
(19) Methyl commate A 38 91.08 01.96
Acetone extract
(20) 9,12-Octadecadienoyl chloride, (Z,Z)- 3 55.93 00.63
(21) Phytol 4 56.15 12.95
(22) Glycerol 1-palmitate 6 68.25 01.73
(23) Di-n-octyl phthalate 7 68.62 00.47
(24) (2,3-Diphenylcyclopropyl) methyl phenyl sulfoxide 8 69.07 00.93
(25) Nerolidol propionate 11 70.88 02.76
(26) Squalene 14 75.454 15.64
(27) Nonacosane 15 77.45 01.01
(28) 2,2,4-Trimethyl-3-(3,8,12,16-tetramethyl-heptadeca-3,7,11,15-tetraenyl)-cyclohexanol 16 78.34 00.86
(29) Hentriacontane 17 81.98 03.37
(30) 𝛽-Amyrin 25 89.50 05.34
Hexane extract
(31) Benzene, 1-methyl-3-(1-methylethyl)- 1 11.91 00.49
(32) 𝛾-Terpinene 2 13.37 01.03
(33) trans Sabinene hydrate 3 14.04 00.66
(34) Carvacrol 4 25.32 37.73
(35) trans-caryophyllene 5 30.40 07.37
(36) 𝛼-Humulene 7 32.03 02.15
(37) Caryophyllene oxide 8 37.59 01.00
(38) Phytol 9 56.13 03.19
(39) Squalene 10 75.43 04.75
(40) Pentacosane 11 81.95 00.86
(41) Stigmasterol 13 86.25 01.29
(42) Tetratriacontane 14 86.57 08.80
(43) Hexatriacontane 16 88.57 01.02
(44) 𝛼-Amyrin 18 91.07 01.53
(45) Hexatriacontane 19 91.33 01.47
(46) Tetrapentacontane 20 92.76 13.77
Evidence-Based Complementary and Alternative Medicine 7

20 peaks, including carvacrol (37.7%), trans-caryophyllene leaves. Because of the lower extractive yield obtained in
(7.3%), squalene (4.7%), tetrapentacontane (13.7%), and tetra- this study, it is recommended to use other better extraction
triacontane (8.8%) as the major components. methods in P. amboinicus. Nevertheless, it is reported that
the biological properties depend not only on the total extract
4. Discussion yield but also on its phytochemical composition [22].
In the human body, cell damage may induce the gener-
Recent years have witnessed the importance of phytochemi- ation of increased levels of free radicals. Various disorders
cals due to their remarkable health benefits. Plant metabolites including myocardial infarction, cancer, atherosclerosis, and
are extracted by using various ways, such as maceration, neurogenerative disorders are mainly correlated to these free
decoction, soxhlet extraction, microwave-assisted extraction, radicals [4]. Antioxidants are chemical compounds and have
supercritical fluid extraction, and ultrasound-assisted extrac- the ability to prevent various oxidative stress related cell dam-
tion method. As maceration technique is a simple and the ages mediated by free radicals. Many antioxidant molecules
easiest method, it is widely used at the preliminary research are recorded in several medicinal plants and thus will be
level [11]. The efficiency of extraction depends on several fac- beneficial in the treatment of several human diseases [15].
tors, including the nature of phytochemical constituents, the Most frequently, the DPPH and FRAP assay methods are pre-
method of extraction, particle size of the sample, extraction ferred for determining antioxidant activity [9]. In this study,
time, temperature, pH, solute to solvent ratio, and the solvent radical scavenging activity was observed to be concentration
polarity [12]. The appropriate use of solvent system is crucial dependent and corroborates with the previous reports on
in order to recover higher extract yield, polyphenols, and several other plant species [4, 9, 23]. The superior radical
some other bioactive compounds from a sample [13]. Plant scavenging potential of plant solvent extracts may be interre-
derived polyphenols including flavonoids possess several lated to the presence of various antioxidants such as polyphe-
biological properties and thus necessitate evaluating their nolic compounds [14, 23]. Even though earlier reports have
presence in different plant parts extracted in different organic stated that P. amboinicus leaf extracts possess antioxidant
solvents [4, 14, 15]. This study revealed the existence of a properties [2], none of them have reported the systematic
significant difference in the extract yield obtained by different study comparing the effect of different solvents on the antioxi-
solvents. Further, the highest extract yields and total phenolic dant potential of P. amboinicus species growing under tropical
content obtained in the methanolic leaf extract of P. amboini- conditions of Malaysia. The methanol extract was found to
cus could be attributed to its high polarity [13]. Another exhibit the highest scavenging activity when compared to
possible reason might be due to the establishment of com- other solvent extracts. This could be attributed to the fact that
plexes by phenolic constituents with other biomolecules such the methanol extract possesses higher antioxidant molecules
as proteins, carbohydrates, terpenes, and inorganic com- such as phenolic compounds as evidenced by our phyto-
pounds that can be more easily recovered from methanol chemical analysis (Table 2). In contrast, Bhatt and Negi [19]
in comparison to other solvents [16]. Similarly, the use of reported that acetone extract of P. amboinicus possessed
methanol as the best solvent for extraction is well docu- superior antioxidant activity when compared to hexane, ethyl
mented in the literature [4, 13, 17]. On the contrary, methanol, acetate, or methanol extracts. According to Rai et al. [24], the
acetone, and water are reported to be ineffective for extracting effective antioxidant activity of P. amboinicus leaves was
polyphenols from the seeds of grapes [18]. In the present observed when extracted with the solvent, ethanol. This
study, the extractive yield and total phenolic content were bet- difference could be could be due to differences in the geo-
ter evidenced in the nonpolar hexane extract when compared graphical growth conditions as stated previously by Swamy
to the polar solvent, acetone. In contrast, the hexane extract et al. [4]. As stated by the earlier reports, the free radical
yielded much lesser total flavonoid content compared to the scavenging activity of different solvent extracts of plants
acetone extract. This difference in the extract yields could mainly depends on the existence of different bioactive chem-
be attributed to the occurrence of a wide range of diverse ical constituents [1, 5]. Further, more polar solvents can often
phytochemical components in P. amboinicus leaves and extract antioxidant compounds in higher quantities. Over-
differences in the solvent polarity. Furthermore, it is reported all, high-polarity solvent (methanol) was very effective in
that the level of solubility of a phenolic compound decides extracting more antioxidant compounds when compared to
the recovery percentage of a particular solvent type [4, 13]. In an intermediate polar solvent, acetone, and nonpolar solvent,
contrast, according to the report by Bhatt and Negi, [19], the hexane.
acetone extract contained significantly higher total phenolics Antimicrobial agents (synthetic or plant based) effectively
compared to the methanol or hexane extracts, which was kill a wide range of infectious microbes. However, their
endorsed for the difference in the geographic location and improper and overuse has permitted microbes to develop
other environmental factors [20]. Likewise, earlier study has multidrug resistance ability which is a major threat and a big-
identified the presence of polyphenols such as caffeic acid, ger challenge to the medical world [25]. Hence, there is a need
coumaric acid, rutin, quercetin, and gallic acid in the acetone to investigate novel molecules to overcome antimicrobial
leaf extract of P. amboinicus [21]. As there is a dearth of resistance. Further, present research emphasizes on the devel-
scientific details on phytochemistry of Malaysian P. amboini- opment of plant based drugs due to the fact that synthetic
cus, this study will be certainly appreciated. In general, this drugs cause several side effects in humans. In this regard, we
study suggests the use of methanol and hexane solvents for evaluated the antimicrobial properties of P. amboinicus leaf
higher recovery of extractable compound from P. amboinicus extracts and the results were very conclusive. All the tested
8 Evidence-Based Complementary and Alternative Medicine

microorganisms were effectively inhibited by all solvent various bioactive compounds in hexane and acetone extracts
extracts with the exception of a gram-negative bacteria E. of P. amboinicus leaves. In addition, compounds in different
coli, which was inhibited only by the methanolic leaf extract. solvent extracts of Malaysian P. amboinicus plants are yet to
MRSA strain exhibited less susceptibility to all solvent be studied. Accordingly, GC-MS analysis was performed in
extracts when compared to other gram-positive strains. B. the preset study and identified a total of 46 compounds from
subtilis was highly susceptible to the methanol extract. Inter- different solvent extracts. The highest number of compounds
estingly, methicillin-resistant S. aureus and C. albicans were (19) was evidenced in the methanolic leaf extract followed by
highly susceptible to all the extracts. Similarly, Gurgel et al. a hexane extract (16) and acetone extract (11). Most of these
[26] reported the efficacy of the hydroalcoholic leaf extracts compounds are known to exhibit various pharmacological
of P. amboinicus from Brazil against MRSA strains while activities. In this study, the higher antioxidant and antimi-
Manjamalai et al. [27] report the anticandidal activity of the crobial activities of the methanol extract could be correlated
methanolic leaf extract of Indian P. amboinicus. To support to the occurrence of more number of bioactive compounds
this, researchers have stated that mostly gram-negative bac- including n-hexadecanoic acid, Phytol, and squalane. Simi-
teria exhibit more resistant properties against a wide range larly, the occurrence of these compounds is also evidenced
of antibiotics or chemical drugs when compared to gram- in many medicinal plants leaves extracted with methanol
positive bacteria [19, 28]. This difference in the susceptibility [5, 33, 34]. Phytol is an important diterpene that possesses
of a microbe to plant extracts is also linked to differences in antimicrobial, antioxidant, and anticancer activities [5, 35,
their cell wall and outer membrane structures [29]. MRSA 36]. Hexadecanoic acid is known to exhibit strong antimi-
cause several human health problems including skin infec- crobial and anti-inflammatory activity [5, 37]. Squalene is
tions, sepsis, pneumonia and other infections, and C. albicans a triterpene that act as natural antioxidants and possess
is a prime causative agent of candidiasis. Thus, P. amboinicus various pharmacological importance [38, 39]. Neophyta-
leaf extracts may be beneficial in treating these diseases diene is a good analgesic, antipyretic, anti-inflammatory,
causing microbes. Microbial inhibition was found to vary antimicrobial, and antioxidant compound [33]. Similarly, the
with the type of solvent extracts evaluated. Likewise, previous compounds 𝛼 and 𝛽-Amyrin present in hexane and methanol
researchers also have testified the wide-ranging antimicrobial extracts are known to possess antidiabetic, anti-inflamma-
activity of this plant [2, 19]. However, these results contradict tory, antiarthritic, and anticancer activities [34]. The presence
with the findings by Bhatt and Negi [19], where the superior of compounds such as carvacrol, caryophyllene oxide, and
antimicrobial activity was shown by the acetone extract, stigmasterol in hexane extract is also reported with various
while Jiyauddin et al. [8] report the highest antimicrobial biological activities [2, 33]. Similarly, Kunle et al. [39] also
activity in the ethanolic leaf extract. This discrepancy could showed the occurrence of carvacrol in the hexane extract
be due to differences in the extraction methods and microbes of Lippia multiflora leaves and showed potent antimicrobial
tested. The highest antimicrobial activity of methanol extract activity [39]. Carvacrol, an isoprenyl phenol is reported as one
against all the examined microorganisms could be due to of the strongest antimicrobial agents [40]. Moreover, it has
more soluble bioactive compounds extracted in the methanol shown to inhibit the biofilms formation by Chromobacterium
solvent. These bioactive compounds, perhaps, may inhibit violaceum, Typhimurium DT104, Salmonella enterica, and
microbial growth effectively by binding to their cell surface. Staphylococcus aureus [41]. The compound, caryophyllene
In support of this, previous studies have stated that the oxide, exhibits a wide range of antimicrobial properties [42].
antimicrobial action of plant extract is presumably related Likewise, a steroid compound was also isolated from the
to the collective effect of phenolic compounds adsorption hexane extract of Hydnophytum formicarum [43]. In the same
onto the cell membrane leading to its disruption and cell study, stigmasterol was shown to possess better antimicro-
leakages and the generation of hydroperoxides by phenolic bial and antioxidative properties. This could be one of the
compounds [2, 30]. These results support the reports empha- reasons for the higher antimicrobial activity exhibited by
sizing on the curative property of P. amboinicus plant extracts hexane extract compared to acetone extract as stated earlier.
and its essential oil [2]. However, most of the research studies However, some of the other major compounds, including
have evaluated antimicrobial activities of essential oil of P. tetracontane, pentacosane, tetratriacontane, tetrapentacon-
amboinicus and limited importance is given on different plant tane, and hentriacontane, are yet to be described in detail.
extracts, especially, in those plants growing under Malaysian Nevertheless, more research efforts are required to isolate,
conditions [2, 8, 20, 26, 27, 31]. Furthermore, this study estab- characterize, and evaluate these compounds from P. amboini-
lishes the importance of the solvents in recovering higher cus leaves to validate their various pharmacological impor-
extractable compounds with potent antimicrobial action tance.
against human pathogens for the first time.
Various biological activities of P. amboinicus leaf extract 5. Conclusion
can be described by investigating the chemical composition
of each extract using GC-MS analysis. The literature sur- In this study, the presence of various soluble bioactive
vey reveals the occurrence of about 30 nonvolatiles from compounds in P. amboinicus leaf extracts greatly contributed
methanol, ethyl acetate, water, and chloroform extracts of P. to antioxidant and antimicrobial activities. Phytochemical
amboinicus leaves, stems, and roots around the world [2, 19, composition and yield of the extract varied depending on
32]. However, to the best of our knowledge, there is no report the solvent types used for the extraction. In the methanolic
of GC-MS based metabolite profiling to detect the presence of extract, more soluble phytocompounds were noticed. The
Evidence-Based Complementary and Alternative Medicine 9

results clearly support the use of P. amboinicus in traditional [10] S. M. Kumara, K. M. Sudipta, P. Lokesh et al., “Phytochemical
medicinal practices to treat various diseases. Thus, this plant screening and in vitro antimicrobial activity of Bougainvil-
can serve as a new natural source for obtaining many lea spectabilis flower extracts,” International Journal of Phy-
therapeutically valued metabolites against various diseases. tomedicine, vol. 4, no. 3, pp. 375–379, 2012.
[11] N. N. Azwanida, “A review on the extraction methods use in
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The authors declare that they have no conflicts of interest. [12] Q. D. Do, A. E. Angkawijaya, P. L. Tran-Nguyen et al., “Effect
of extraction solvent on total phenol content, total flavonoid
content, and antioxidant activity of Limnophila aromatica,”
Acknowledgments Journal of Food and Drug Analysis, vol. 22, no. 3, pp. 296–302,
2014.
The authors sincerely thank Department of Crop Science and
Laboratory of Natural Products, Universiti Putra Malaysia, [13] S. B. Iloki-Assanga, L. M. Lewis-Luján, C. L. Lara-Espinoza et
for providing laboratory facilities. al., “Solvent effects on phytochemical constituent profiles and
antioxidant activities, using four different extraction formula-
tions for analysis of Bucida buceras L. and Phoradendron cali-
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