Effectiveness of Zingiberaceae Herbal Extracts

Orawan Thipmanee, et al.

Articles

MOLEKUL
eISSN: 2503-0310

https://doi.org/10.20884/1.jm.2025.20.2.14695

Effectiveness of Zingiberaceae Herbal Extracts from the Hala-Bala Forest for Application
in Thai Massage
Orawan Thipmanee1, Sunee Waema1, Saluma Samanman2, Aeesoh Benhawan1*
Major of Chemistry, Faculty of Science Technology and Agriculture, Yala Rajabhat University,
Yala 95000, Thailand
2
Faculty of Science and Technology, Princess of Naradhiwas University, Narathiwat 96000, Thailand
1

*Corresponding author email: aeesoh.b@yru.ac.th
Received February 07, 2025; Accepted April 22, 2025; Available online July 20, 2025
ABSTRACT. This research aimed to study the efficiency of essential oils extracted from Zingiberaceae herbals that are
Etlingera elatior, Zingiber montanum, and Etlingera coccinea, collected from the Hala-Bala forest in Chulabhorn Pattana
Village 9, Ban Santisuk 2, Mae Wat Sub-district, Than To and Betong District, Yala Province. The essential oils were extracted
using steam distillation and characterized based on their physical and chemical properties, antimicrobial activity,
phytochemical composition, and antioxidant activities. The pH values of the essential oils were found to be 6.1, 4.9, and
6.2, respectively. Heavy metal analysis of the essential oils, conducted using a flame atomic absorption spectrophotometer,
revealed no contamination with lead, chromium, manganese, and cadmium. In antimicrobial activity test against three
bacterial strains— Staphylococcus aureus, Bacillus spp., and Escherichia coli—the essential oils of E. elatior and Z.
montanum demonstrated inhibitory effects against all three strains, with inhibition zones of 12.30.28, 10.71.06, and
11.21.01 mm for E. elatior and 22.11.25, 26.00.70, and 18.50.70 mm for Z. montanum. Meanwhile, the essential
oil of E. coccinea inhibited S. aureus and E. coli with inhibition zones of 20.50.70 and 13.20.35 mm, respectively.
Preliminary phytochemical analysis was performed using high-performance thin-layer chromatography (HPTLC), and
antioxidant activity evaluated using three assays: DPPH radical scavenging activity (DPPH assay), ABTS free radical bleaching
(ABTS assay), and ferric reduction of antioxidant power (FRAP assay). The results confirmed that all three essential oils
exhibited antioxidant activity, and the phytochemical screening detected flavonoids, diterpenes, and anthraquinones. This
research highlights the potential of the essential oils for developing Thai massage health products, particularly in the form
of massage oils infused with natural extracts and local herbs. Volunteer satisfaction assessments conducted at the Thai
Traditional Medicine Learning Center, Yala Rajabhat University, Yala Province, Thailand, indicated a high to the highest
level of satisfaction. This was attributed to the oils' natural composition, low toxicity, and high effectiveness.
Keywords: Antibacterial, Antioxidant, Etlingera elatior, Essential oil, Etlingera coccinea, Phytochemical, Zingiber montanum

INTRODUCTION
Plants with aromatic and medicinal properties have
been used for various purposes, including adding
flavor to food (Saffarionpour, 2024), medicinal
applications (Parvin et al., 2023), preservation (Liu et
al., 2024), beauty care (Anuradha and Bharadvaja,
2023), and stress relief products (Kate et al., 2023).
These plants serve as a promising natural alternative,
offering numerous benefits, safety, and sustainability.
The therapeutic use of medicinal plants dates back to
the earliest stages of human civilization. The herbal
system of medicine is not only the oldest form of health
care but also an essential component of modern
civilization’s development. Even today, a vast majority
of the global population, particularly in the developing
countries, continues to rely on herbal medicine and its
products for primary health care needs.
Plants from the Zingiberaceae family, commonly
known as ginger, are well-known for their strong

aromatic and medicinal properties (Wahyuni et al.,
2023). These plants are widely distributed throughout
the tropics, particularly in Southeast Asia, and consists
of 53 genera and over 1300 species (Pandey et al.,
2023). The Zingiberaceae plants are commonly used
in traditional medicine due to its various
pharmacological and physiological effects. Studies
have shown that their rhizomes are effective in treating
several medical conditions, including digestive,
respiratory, nervous, and muscular system problems,
as well as degenerative diseases (Zulfadhly et al.,
2023). The plant is used to treat a wide range of
ailments such as nausea, vomiting, epilepsy, sore
throat, coughs, colds, bruises, wounds, liver
complaints,
rheumatism,
muscle
aches,
atherosclerosis, migraine headaches, high cholesterol,
ulcers, and stomach discomfort (Pham et al., 2021).
Phenolic compounds, secondary metabolites found in
the rhizomes of Zingiberaceae plants, have been

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shown to possess significant biological activity that
benefits human health and have antibacterial
properties useful in food and pharmaceutical
applications (Alfuraydi et al., 2024; Mutakin et al.,
2023; Rungruang et al., 2021). Zingiberaceae plants
are rich in essential oils (EOs), which have important
functions in nature and are commercially used in
industries, including flavors, fragrances, cosmetics,
agriculture, pharmaceuticals, health-care products,
and others (Ivanovi´ et al., 2021). The primary
objective is to replace synthetic products with natural
EOs, which offer health benefits and also reduce
adverse environmental impacts. Certain EOs have
been found to possess potent antimicrobial,
preservative, herbicidal, and antioxidant properties
beneficial for many industries (Amil et al., 2024;
Rawat et al., 2024; Vaishnavi et al., 2024).
In Thailand, the Hala-Bala forest complex is
primarily tropical rainforest, with vegetation similar to
that of northern Malaysia, which it borders. The
species found in the southernmost provinces of
Thailand's Hala-Bala forest, including Etlingera elatior
(E. elatior), Zingiber montanum (Z. montanum), and
Etlingera coccinea (E. coccinea) are strongly aromatic
herbs with medicinal properties. However, there are
very few studies on the extraction of EOs from plants
in the Hala-Bala forest area. Therefore, this presents
an interesting and challenging opportunity for
researchers in the region.
Most of the previously reported studies have shown
that Zingiberaceae plants and their derivatives may be
responsible for the antioxidant potential and
antibacterial activity of ginger EO (Alfuraydi et al.,
2024; Badrunanto et al., 2024; Gunasena et al.,
2022; Mutlu-Ingok et al., 2021). However, the
previous studies were limited to investigation of E.
elatior, Z. montanum, and E. coccinea from the HalaBala forest, Thailand. Therefore, this research focuses
on the effectiveness of EOs from Zingiberaceae plants,
specifically E. elatior, Z. montanum, and E. coccinea
from the Hala-Bala forest area in Chulabhorn Pattana
Village 9, Ban Santisuk 2, Mae Wat Sub-district, Than
To and Betong District, Yala Province. The physical
and chemical properties of the EOs were determined,
along with analysis of phytochemical compounds,
antimicrobial activity and antioxidant properties.
These EOs have also been developed into health
products with traditional Thai massage in the form of
massage oils containing EOs and oils from local
herbs. This helps increase the value of local resources
and provides another option for incorporating local
herbs into traditional Thai medicine.
EXPERIMENTAL SECTION
Materials
Plant materials were collected from the Hala-Bala
forest in Chulabhorn Pattana Village 9, Ban Santisuk
2, Mae Wat Sub-district, Than To and Betong Districts,
Yala Province, Thailand. The rhizomes were separated

from the plants, thoroughly cleaned, and prepared for
use. Alpha-pinene, eucalyptol, and terpininen-4-ol
were obtained from Sigma-Aldrich, USA. Nutrient
agar and nutrient broth were purchased from
Himedia, India.
Extraction of Essential Oils from E. elatior, Z.
montanum, and E. coccinea
The extraction of EOs was studied using two
methods: steam distillation — following a modified
method described by Wainer et al., (2022) and boiling
in coconut oil with steam, due to their simplicity and
short extraction time. Moreover, these methods allow
for the use of a large sample quantities, resulting in a
higher yield of EO.
For the steam distillation method, the rhizomes of
E. elatior, Z. montanum, and E. coccinea were peeled,
thoroughly washed, and then cut into small pieces (1
 1 cm). These pieces were subsequently dried for
three hours. Afterward, 75 g of each rhizome sample
was placed in a round-bottom flask and distilled with
500 mL of distilled water. The water was heated until
it reached boiling point, producing steam, which rose
and passed through the fresh rhizomes. As the steam
carried the EOs through the system, it was directed into
a condenser, where it cools and condensed back into
liquid form. The EO layer was then carefully
separated, transferred into an opaque vial, and stored
at 4°C. The total volume of EOs extracted was
measured and recorded. For the second method,
freshly cut rhizome pieces were mixed with coconut oil
at a 1:1 (% v/v) ratio and subjected to steam distillation
for 30 minutes. Following the distillation process, the
EOs were separated using a syringe, then stored in an
opaque vial at 4 C to maintain their stability and
prevent degradation.
The pH values of the extracted EOs were
determined using a pH meter (SI Analysis, Germany)
which was calibrated with pH standard buffers
before use to ensure accuracy. Additionally, the
presence of heavy metals, including lead (Pb),
cadmium (Cd), chromium (Cr), and manganese (Mn),
was analyzed using a flame atomic absorption
spectrophotometer (Flame-AAS) (Shimadzu, Japan,
AA 7000). The specific wavelengths used for detection
were 283.3 nm for Pb, 228.8 nm for Cd, 367.9 nm
for Cr, and 279.5 nm for Mn.
EO samples were digested using the wet digestion
method. In a 100 mL beaker, 10 ml of nitric acid was
added to 15 mL of EO sample. After digestion, the
mixture was allowed to cooled, and 3 mL of 30%
hydrogen peroxide was added. The beaker was
covered, and the sample was gently heated to initiate
the peroxide reaction. Subsequently, 5 mL of
concentrated hydrochloric acid and 10 mL of
deionized water were added, and the sample was
further heated for 15 minutes without boiling. After
cooling, the sample was filtered through a Whatman
No. 5 filter paper and diluted to a final volume of
50 mL with deionized water.

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Orawan Thipmanee, et al.

Extraction of Essential Oils from Citronella Grass for
Use as an Ingredient in Massage Oil Formulations
Citronella oil stimulates the muscles, nervous
system, tissues, and skin, while helping to reduce
aches and pains. It promotes blood circulation and
can alleviate muscle spasms, back pain, sprains and
cramps, as well as provide relief from fatigue or
headaches. Therefore, it is used as an ingredient in
herbal massage oil products. For EO extraction,
washed samples and further chopped into small
pieces and dried for 1 hour. Then 75 g of citronella
and 150 mL of distilled water were distilled using the
steam distillation method. After thorough distillation,
the citronella EO was collected in an opaque vial and
stored at 4 °C.
Antibacterial Testing
Antibacterial testing was performed following to
the method described by Santoni et al., (2024). Three
pathogenic bacteria strains were selected for this study
to assess antimicrobial activities: S. aureus, Bacillus
spp., and E. coli. These pathogens were obtained from
the Medical and Industrial Microbiology program
atYala Rajabhat University, Thailand. All bacterial
cultures were maintained on nutrient agar (NA).
Bacteria colonies were transferred into 5 mL of
nutrient broth (NB) and incubated at 37 oC for18-24
hours. The inocula were standardized with sterile NB
to achieve a final cell concentration of 106-107
CFU/ml. The disc diffusion test was performed as
described by Shimanuki and Knox (2000). An
inoculum suspension of each bacterial strain was
swabbed onto NA plates. Sterile 6 mm Whatman No.
1 filter paper discs were aseptically placed on plates.
Then, 40 µL of volatile EOs were applied to the surface
of the Whatman paper discs, which were cut into
circles with a diameter of 6 mm for assay activity. After
incubating the plates for 24-48 hours at 35-37 oC, the
diameter of the inhibition zone was measured as an
indicator of bacterial inhibition.

Visible spectrophotometer. The percentage of DPPH
inhibition was calculated and expressed as
micrograms of Trolox-equivalent antioxidant capacity
(µg TEAC) per milliliter of sample.
% Inhibition = [(A control – A sample)/A control] × 100
Antioxidant Activity Analysis Using the ABTS Radical
Cation Decolorization Assay
A standard curve of Trolox in ethanol was prepared
using different concentrations (10, 20, 30, 40, and 50
µg/mL). The ABTS radical (2,2-azino-bis(3ethylbenzthiazoline-6-sulphonic acid)) was generated
by mixing a 7 mM ABTS solution in ethanol (50 mL)
with a 2.45 mM potassium persulfate solution in
ethanol (25 mL) at a volume ratio of 2:1. The mixture
was stored in the dark at room temperature for 16
hours. Subsequently, the EO extract was mixed with
the ABTS solution at a volume ratio of 1:2 and left in
the dark at room temperature for 5 min. The
absorbance was then measured at a wavelength of
734 nm using a UV-Visible spectrophotometer. The
percentage of ABTS•+ radical inhibition was
calculated, and the results were expressed as
micrograms of Trolox-equivalent antioxidant capacity
per milliliter of sample.
%ABTS•⁺ Radical Inhibition = [(A control – A test sample)/

Phytochemical Preliminary Test
The qualitative phytochemical screening test of EO
and herbal massage oil identified the main classes of
active components —flavonoids, tannins, diterpenes,
and anthraquinones — following a modified method
described by Shaikh and Patil (2020).

A control] × 100
Antioxidant Activity Analysis Using Ferric Ion Reducing
Antioxidant Power (FRAP) Assay
The FRAP reagent was prepared by mixing an
acetate buffer (pH 3.6) with a 20 mM ferric chloride
solution (FeCl3·6H2O) and a 10 mM TPTZ (2,4,6tripyridyl-s-triazine) solution in 40 mM hydrochloric
acid at a ratio of 10:1:1, respectively. Then, EO extract
was then mixed with the FRAP reagent according to the
details in Table 5. The mixture was shaken thoroughly
and allowed to stand for 4 minutes before measuring
the absorbance at a wavelength of 593 nm. The
absorbance value was calculated using the equation:
Absorbance = Atest sample – Ablank – Acontrol.
The electron-donating capacity was determined by
comparing the result to a standard curve of ferrous
sulfate. The results were expressed as mM Fe2+
equivalents per gram of sample. (Benzie & Strain,
1996; Pulido et al., 2000).

The DPPH Assay for Radical Scavenging Activity was
Performed Following to the Method Described by of
Eugenio José Garcia et al. (2012).
A standard curve of Trolox in ethanol was prepared
using various concentrations (10, 20, 30, 40, and 50
µM/mL), and the DPPH inhibition percentage for each
concentration was calculated. A 0.5 mM solution of
2,2-diphenyl-1-picrylhydrazyl (DPPH) was prepared in
500 mL of ethanol. The EO extract was then mixed
with the DPPH solution at a 9:1 volume ratio,
thoroughly shaken, and kept in the dark at room
temperature for 30 minutes. After incubation,
absorbance was measured at 517 nm using a UV-

HPTLC Analysis of Plant Extract
In the HPTLC analysis of plant extracts, a
quantitative assessment of alpha-pinene, eucalyptol,
and terpinen-4-ol was performed under specific
chromatographic conditions. Standard solutions of
alpha-pinene, eucalyptol, and terpinen-4-ol were
prepared by dissolving 1 mg of each compound in 10
mL of methanol. Sample solutions, including EOs and
herbal massage oil preparations, were prepared by
dissolving 1 g of each sample in 10 mL of methanol.
For the stationary phase, precoated silica gel 60 F254
plates (20 x 10 cm, Merck, Darmstadt, Germany) were
used. The mobile phase for alpha-pinene and

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eucalyptol consisted of a solvent mixture of n-hexane
and ethyl acetate in a volume ratio of 8:2, while for
terpinen-4-ol, a combination of toluene, ethyl acetate,
and glacial acetic acid in a ratio of 8:2:0.3 was
employed.
Sample application was carried out using a
CAMAG Linomat-5, with 15 tracks per plate. Each
band was 8 mm long, spaced 11.4 mm apart,
positioned 8 mm from the lower edge, and the first
application starting 20 mm from the left edge.
Chromatogram development was conducted in an
automatic developing chamber (CAMAG ADC-2) with
chamber saturation for 20 minutes and conditioning
at 33% relative humidity for 10 minutes using a
saturated magnesium chloride solution. Plates were
developed to a migration distance of 70 mm and dried
for 5 minutes. Derivatization involved treatment with
anisaldehyde-sulfuric acid reagent, followed by
heating at 100˚C for 5 minutes.
Quantitative evaluation of active compounds in
EOs and herbal massage oils was performed using the
CAMAG TLC Scanner 4. The concentrations of alphapinene, eucalyptol, and terpinen-4-ol in samples were
determined by comparing the peak height or area of
the chromatograms to reference standards.
Calibration plots were generated by applying various
volumes of standard solutions (1, 2, 3, 4, 5, 6, and 7
µL). Chromatographic analysis was conducted at a
wavelength of 500 nm, and standard curves
illustrating the relationship between the area under the
curve and concentration were constructed. The linear
relationship was expressed using the equation y =
slope(x) + intercept, and the quantification of active
compounds was performed using winCATS 1.2.6
software.
Preparation of Herbal Massage Oil Products
The incorporation of EOs and other herb
components into herbal massage oil formulations has
gained increasing popularity in recent years (Meha et
al., 2024; Ningsih et al., 2023; Vora et al., 2024).
This study investigated three herbal massage oil
products formulated with EOs from E. elatior, Z.
montanum, and E. coccinea together with other
ingredients. Virgin coconut oil (100 mL) was measured

using a digital scale and mixed menthol (100  10 g),
patchouli (75  7.5 g), camphor (75  7.5 g),
wintergreen oil (50  5 g), phlai oil (50  5 g), turmeric
oil (50  5 g), and EOs (50  5 g) from E. elatior, Z.
montanum, and E. coccinea. The mixture was stirred
until homogeneous using an overhead stirrer. The
resulting massage oils were then stored in tightly
sealed containers and subjected to a consumer
preference testing.
RESULTS AND DISCUSSION
Extraction of the Essential Oils
In this work, two different methods—steam
distillation and boiling in coconut oil with steam—were
used to extract EOs from the rhizomes of E. elatior, Z.
montanum, and E. coccinea. The results showed that
the steam distillation method (Figure 1) yielded 5 mL
of EO within 4 hours, which was 10 times higher than
the yield obtained from the boiling method (0.5 mL).
This is because plant materials are directly exposed to
steam, which facilitates the release of EOs through
evaporation. Additionally, steam distillation offers
advantages such as simplicity and low cost, making it
a more efficient extraction method.
Characterization of Essential Oils
pH Determination
pH can be defined as the amount of free
hydronium ions in a solution and represents active
acidity. The pH value of the EOs from E. elatior, Z.
montanum, and E. coccinea were measured as 6.10
 0.05, 5.10  0.11, and 6.20  0.07, respectively.
The differences in pH values of the EOs from the three
Zingiberaceae herbs, especially the pH of Z.
montanum, are due to variations in soil conditions
suitable for their cultivation and growth. The optimal
pH range for growing E. elatior and E. coccinea is
between 6.0 and 7.0, while Z. montanum thrives in
soil with a pH of 5.5–6.5. Therefore, soil conditions in
different areas are key factors influencing the pH
values of the EOs. However, all their pH values fall
within the accepted standard range of the Industrial
Product Standard that is set by ISO 1242:1999 (E). It
was shown that the EOs were slightly acidic that can
be used safely.

Figure 1. A typical steam distillation unit for extraction of the essential oils.

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Orawan Thipmanee, et al.

Heavy Metal Analysis
Herbal EOs from E. elatior, Z. montanum, and E.
coccinea were analyzed using the wet digestion
method. The results showed that none of the four
heavy metals, namely Pb, Cd, Cr, and Mn were
detected. During distillation in primitive stills or storage
in opaque vials, metal impurities could not be released
into EOs.
Antimicrobial Activity of Essential Oils
The ability of EOs from E. elatior, Z. montanum,
and E. coccinea to inhibit bacteria was tested, as
shown in Table 1. The inhibition of three bacterial
strains—S. aureus, Bacillus spp., and E. coli— was
evaluated using disc diffusion method. Z. montanum
exhibited the best inhibition against all three species of
pathogenic bacteria, with inhibition zones of
22.1±1.25, 26.0±0.70, and 18.5±0.70 mm,
respectively. E. elatior showed inhibition against all
three species, with inhibition zones of 12.3±0.28,
10.7±1.06, and 11.2±1.01 mm. E. coccinea
inhibited two species of pathogenic bacteria with
inhibition zones of 20.5±0.70 and 13.2±0.35 mm,
respectively.
Antimicrobial Activity of Herbal Massage Essential Oil
Products
The antibacterial efficiency of herbal massage oil
products derived from E. elatior, Z. montanum and E.
coccinea was evaluated against three bacterial strains:
S. aureus, Bacillus spp., and E. coli using the disc
diffusion method. The results showed that the
inhibition zones for S. aureus were 31.8±2.3,
33.5±1.0, and 33.2±0.3 mm, respectively. The
inhibition zones for Bacillus spp. were 28.0±0.7,
30.1±0.5, and 29.5±2.2 mm, and for E. coli, the
zones were 39.1±0.05, 40.3±0.1, and 39.6±0.2
mm. These results indicate that the herbal massage
oils from E. elatior, Z. montanum, and E. coccinea are

capable of inhibiting the growth of all three bacterial
strains. Among these, the herbal massage oil derived
from Z. montanum exhibited the highest antibacterial
efficiency, followed by E. elatior and E. coccinea,
respectively, as in Table 2. This is because its EO has
a higher acidity level compared to the other two EOs,
making it more effective in inhibiting bacterial growth.
Additionally, Z. montanum contains a greater number
of bioactive chemical compounds that contribute to its
antibacterial properties compared to E. elatior and E.

coccinea.

Preliminary Phytoconstituent Analysis
The preliminary phytoconstituent analysis of EOs
and herbal massage oils from Etlingera and Zingiber
species revealed both similarities and differences in
bioactive compounds. As shown in Table 3, the EOs of
E. elatior, Z. montanum, and E. coccinea contained
flavonoids and diterpenes, which are known for their
antioxidant and anti-inflammatory properties (Liga et
al., 2023; Câmara et al., 2024; Vo et al., 2020). The
herbal massage oils containing E. elatior, Z.
montanum, and E. coccinea exhibited additional
secondary metabolites, including tannins and
anthraquinones. The EOs added to the herbal
massage oils enhance the therapeutic potential, as
flavonoids play a key role in relieving pain, chronic
pain, and soreness associated with conditions like
arthritis or muscle fatigue (Scuteri et al., 2021; Bako
et al., 2023; Al-Khayri et al., 2022). In addition,
diterpenes help reduce swelling, inflammation, and
discomfort in muscles and joints. Their neuroprotective
properties may contribute to managing nerve-related
pain (Del Prado-Audelo et al., 2021; Habtemariam,
2023; Al-Khazaleh et al., 2024). Together, flavonoids
and diterpenes offer a natural and holistic approach
to managing pain, making them valuable components
in traditional and modern therapeutic practices.

Table 1 The inhibition zone of Zingiberaceae herbal extracts (E. elatior, Z. montanum, E. coccinea)
on pathogenic bacteria.
Pathogenic
bacteria

Inhibition zone (mm.)

E. elatior

S. aureus
Bacillus spp.
E. coli

12.3±0.28
10.7±1.06
11.2±1.01
*ND = Not detectable

Z. montanum

E. coccinea

Control
(70% Ethanol)

22.1±1.25
26.0±0.70
18.5±0.70

20.5±0.70
ND
13.2±0.35

30.7±3.18
21.2±0.28
22.8±1.04

Table 2 Inhibition zone of herbal massage essential oils products from E. elatior, Z. montanum, and
E. coccinea on pathogenic bacteria
Pathogenic
bacteria

Inhibition zone (mm.)

E. elatior

Z. montanum

E. coccinea

S. aureus
Bacillus spp.
E. coli

31.8±2.3
28.0±0.7
39.1±0.05

33.5±0.3
30.1±0.5
40.3±0.1

33.2±0.3
29.5±2.2
39.6±0.2

343

Control
(70% Ethanol)
22.5±0.8
18.8±2.7
17.5±1.8

Molekul, Vol. 20. No. 2, July 2025: 339 – 348
Table 3 Preliminary phytoconstituent analysis
Secondary
Metabolites

Essential oils

E.
elatior

Z.
montanum

E.
coccinea

flavonoid

+

+

tannins

-

diterpenes
Anthraquinones

Herbal massage oil products

E.
elatior

Z.
montanum

E.
coccinea

+

+

+

+

-

-

+

+

+

+

+

+

+

+

+

-

-

-

+

+

+

Test
Alkaline
reagent test
Braymer’s
test
Copper
acetate test
Borntrager’
s test

Table 4 Results of linear regression analysis for the calibration of alpha-pinene, eucalyptol, and terpinen-4-ol
Standard
substance
Alpha-pinene

Mobile phase

Rf

n-hexane : ethyl
acetate (8:2)

Eucalyptol
Terpinen-4-ol

toluene : ethyl
acetate : glacial
acetic acid (8:2:0.3)

Regression equation

0.19

Linearity range
(ng/band)
100 - 700

0.52

100 - 700

0.25

100 - 700

y = 11.075x +
1756
y = 16.689x +
487.69

Antioxidant Activity Assays and HPTLC Analysis of
Active Compounds.
The linearity of the three standards was observed
within the range of 100–700 ng/band (Table 4). The
peak areas for seven concentrations of alpha-pinene,
eucalyptol, and terpinen-4-ol were calculated, and
individual calibration curves were plotted for each
compound by concentration versus peak area. The
regression equations for alpha-pinene, eucalyptol,
and terpinen-4-ol were determined to be y = 0.8464x
+ 456.64, y = 11.075x + 1756, and y = 16.689x +
487.69. The correlation coefficients (r) were 0.9977,
0.9902, and 0.9922, indicating strong linearity for
each compound.
The DPPH, ABTS, and FRAP assays provided
insights into the antioxidant capacities of EOs from
Etlingera and Zingiber species, as well as their
respective herbal massage oil formulations. From
Table 5, for the DPPH assay, which measures free
radical scavenging, E. coccinea displayed the highest
antioxidant capacity (556.44 µg Trolox/mL), slightly
higher than Z. montanum (551.47 µg Trolox/mL) and
E. elatior (547.77 µg Trolox/mL). When EOs are
added to a massage oil formula, the antioxidant
activity was significantly enhanced, indicating the
contribution of EOs to the overall antioxidant potential.
The ABTS assay, which also evaluates free radical
inhibition, showed Z. montanum to having the highest
inhibition (585.31 µg Trolox/mL), followed by E.
elatior (382.41 µg Trolox/mL) and E. coccinea
(375.29 µg Trolox/mL). These results align with the
DPPH assays, underscoring the strong antioxidant

y = 0.8464x +
456.64

Correlation
coefficient
0.9977

0.9902
0.9922

properties of Z. montanum. When these EOs were
incorporated into massage oils, Z. montanum
maintained the highest inhibition (525.11 µg
Trolox/mL), followed by E. elatior (458.51 µg
Trolox/mL) and E. coccinea (436.39 µg Trolox/mL).
This suggests that Z. montanum’s robustness as an
antioxidant remains even in diluted or mixed
formulations. In the FRAP assay, which measures the
reducing power (ability to donate electrons), Z.
montanum displayed a notably high antioxidant value
(79,505.88 µg Fe2+/g), far exceeding E. elatior
(1,905.88 µg Fe2+/g) and E. coccinea (1,305.88 µg
Fe2+/g). When added to herbal massage oils, the FRAP
values of all formulations increased, with Z.
montanum again leading (179,141.2 µg Fe2+/g),
followed by E. elatior (157,611.8 µg Fe2+/g) and E.
coccinea (90,670.59 µg Fe2+/g). However, the base
oil without EOs exhibited a substantial FRAP value
(67,131.89 µg Fe2+/g) because the massage oil
formula contains various herbal ingredients. All assays
demonstrated that Z. montanum exhibited the
strongest antioxidant potential in both pure EOs and
those incorporated into massage oil formulations,
particularly in the FRAP assay, with its exceptionally
high reducing power. Both the DPPH and ABTS assays
confirmed that Z. montanum and E. coccinea have
potent free radical inhibition capabilities, with Z.
montanum maintaining its antioxidant capacity even
when added to a massage oil formulation.
The HPTLC analysis of EOs and herbal massage
oils from Etlingera and Zingiber species revealed
the presence of alpha-pinene, eucalyptol, and

344

Effectiveness of Zingiberaceae Herbal Extracts

Orawan Thipmanee, et al.

terpinen-4-ol. In the EOs, E. elatior contained alphapinene at 589.76 ng (0.122%) and eucalyptol at
425.65 ng (0.089%). Z. montanum had alpha-pinene
at 434.56 ng (0.091%), eucalyptol at 432.56 ng
(0.090%), and terpinen-4-ol at 246.39 ng (0.061%).
E. coccinea showed alpha-pinene at 424.94 ng
(0.082%), eucalyptol at 241.56 ng (0.059%), and
terpinen-4-ol at 239.67 ng (0.055%). In herbal
massage oils with EOs added, the levels of these
compounds generally increased. For E. elatior, alphapinene was measured at 611.56 ng (0.086%),
eucalyptol at 441.56 ng (0.065%), and terpinen-4-ol
at 145.56 ng (0.019%). Z. montanum exhibited
increased yields with alpha-pinene at 678.56 ng
(0.092%), eucalyptol at 616.64 ng (0.088%), and
terpinen-4-ol at 345.67 ng (0.051%). E. coccinea
contained alpha-pinene at 638.56 ng (0.090%),
eucalyptol at 412.57 ng (0.067%), and terpinen-4-ol
at 422.65 ng (0.069%). In the herbal massage oil
without added EOs, lower levels of these compounds
were detected: alpha-pinene at 231.46 ng (0.039%),
eucalyptol at 249.78 ng (0.042%), and terpinen-4-ol
at 145.85 ng (0.020%). The HPTLC analysis provides
an understanding of the presence and concentrations
of specific bioactive compounds (alpha-pinene,
eucalyptol, and terpinen-4-ol) in EOs and herbal
massage oils derived from Etlingera and Zingiber
species.
Each component has documented
therapeutic properties: alpha-pinene is known for
its anti-inflammatory and bronchodilator effects
(Salehi et al., 2019), eucalyptol for its potential as
an anti-inflammatory and antimicrobial agent (Hoch
et al., 2023), and terpinen-4-ol for its antioxidant
and antimicrobial benefits (Badr, et al., 2023).
The antioxidant mechanism of alpha-pinene and

eucalyptol involves stabilizing and preventing the
further oxidation of unstable free radicals, such as
hydroxyl radicals (•OH), superoxide anions (O₂•⁻),
and peroxyl radicals (ROO•), by donating hydrogen
atoms (H•). (Xu et al., 2019). In EOs, Z. montanum
displayed the highest concentrations across all
compounds, especially in alpha-pinene (434.56 ng)
and eucalyptol (432.56 ng), suggesting strong
antioxidant and antimicrobial potentials. E. elatior and
E. coccinea also showed significant yields, although
lower than Z. montanum. When incorporated into
massage oils, the levels of these components
increased substantially across all three species, likely
due to the presence of EOs. Specifically, Z. montanum
in the massage oil formulation retained the highest
concentrations, with alpha-pinene reaching 678.56
ng and eucalyptol at 616.64 ng.
Characteristics and Volunteer Satisfaction Test Used of
the Herbal Massage Oil
The observation of the color parameter showed a
yellow result, as displayed in Figure 2. This is due to
the use of Phlai and turmeric oil, which contains
curcuminoids that can give rise to a yellow-lemon
color in the formula. All the herbal massage oils were
clear and in the form of homogeneous liquids, due to
the good solubility of the EOs and the carrier oil, virgin
coconut oil. The good solubility is attributed to the
similar polarity of the oils. In testing the efficiency and
satisfaction of volunteers at the Thai massage clinic,
Yala Rajabhat University, Thailand, who tested three
of herbal massage oils, it was found that all 33
volunteers had satisfaction levels from herbal massage
oil from E. elatior, Z. montanum and E. coccinea EO
were 4.52  0.63, 4.60  0.57 and 4.40  0.65,
respectively. The highest level of overall satisfaction

Table 5 Results of antioxidant activity assays and HPTLC analysis of active compounds.
Antioxidant activity assay
DPPH
ABTS
(µg
(µg Trolox/
Trolox/mL) mL)

FRAP value
(µg Fe2+/g)

E. elatior

547.77

382.41

1,905.88

Z. montanum

551.47

585.31

79,505.88

E. coccinea

556.44

375.29

1,305.88

Sample

HPTLC method
alpha
- Eucalyptol
pinene
(ng, % yield)
(ng, % yield)

Terpinen-4ol
(ng, % yield)

Essential oils
589.76
(0.122)
434.56
(0.091)
424.94
(0.082)

425.65 (0.089)
432.56 (0.090)
241.56 (0.059)

246.39
(0.061)
239.67
(0.055)

Herbal massage oil products

E. elatior

382.76

458.51

157,611.8

Z. montanum

559.60

525.11

179,141.2

E. coccinea

426.53

436.39

90,670.59

No add

328.24

323.10

67,131.89

345

611.56
(0.086)
678.56
(0.092)
638.56
(0.090)
231.46
(0.039)

441.56 (0.065)
616.64 (0.088)
412.57 (0.067)
249.78 (0.042)

145.56
(0.019)
345.67
(0.051)
422.65
(0.069)
145.85
(0.020)

Molekul, Vol. 20. No. 2, July 2025: 339 – 348

(A)

(B)

(C)

Figure 2. Herbal massage oil containing the EOs of E. elatior (A), Z. montanum (B) and E. coccinea (C)
with the massage oil made from Z. montanum EOs,
followed by E. elatior and E. coccinea EOs,
respectively. This is because when aroma massage is
applied to the skin and absorbed into the bloodstream
through the skin pores, it provides a feeling of comfort
and reduces the need for invasive methods of pain
relief. Moreover, massage therapy using EOs is easier,
cheaper, and has no side effects.
CONCLUSIONS
The Zingiberaceae plants also contain valuable
EOs that are useful in various fields, primarily due to
their strong odor and the wide range of
pharmacological effects they possess. The main
constituents of EOs are diterpenes, which exhibit
pronounced antibacterial and antioxidant effects. In
this study, we successfully isolated the EOs of three
plant species: E. elatior, Z. montanum, and E.
coccinea using the green distillation method. The
results confirmed that the antimicrobial effect was
observed with inhibition zones of 22.1±1.25,
26.0±0.70, and 18.5±0.70 mm against three
strains: S. aureus, Bacillus spp., and E. coli. Regarding
antioxidant capacity, alpha-pinene, eucalyptol and
terpinen-4-ol were the main diterpenes present in all
the EOs studied, and these compounds also exhibit
therapeutic properties. Additionally, the application of
massage oils containing EOs was found to be highly
effective and was considered safe for customers, as
they were free from toxic substances. Herbal massage
oil from E. montanum is the most suitable use for the
massage oil. Recommendations for future work
include analyzing the chemical composition of
essential oils (EOs) using the GC-MS technique.
ACKNOWLEDGMENTS
The authors are grateful to Grants from Basic
Research Fund: Fiscal Year 2022 (FF) and also thank
the Chemistry’s Department, Faculty of Science

Technology and Agriculture, Yala Rajabhat University,
Thailand, for providing experimental instruments and
facilities.
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