Effect of Organic Solvents in the Preparation

Sri Rahayu Lestari, et al.

Articles

MOLEKUL
eISSN: 2503-0310

https://doi.org/10.20884/1.jm.2025.20.2.13310

Effect of Organic Solvents in the Preparation of Single Aged Garlic Transfersomes
and Their Phytochemical Activities
Sri Rahayu Lestari1*, Abdul Gofur1, Yunita Rakhmawati1, Suharti2, Nik Ahmad Nizam Nik Malek3,
Dewi Sekar Miasih1, Alif Rosyidah El Baroroh1, Yuslinda Annisa4
Department of Biology, Faculty of Mathematics and Natural Science, Universitas Negeri Malang,
Malang 65145, Indonesia
2
Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Malang,
Malang 65145, Indonesia
3
Centre of Sustainable Nanomaterials, Department of Biosciences, Faculty of Biosciences,
University Teknologi Malaysia, 81310, Malaysia
4
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Brawijaya,
Malang 65145, Indonesia
1

*Corresponding author email : srirahayulestari@um.ac.id
Received October 10, 2024; Accepted May 26, 2025; Available online July 20, 2025
ABSTRACT. Single Aged Garlic (SAG), a fermented product from a single garlic, has been extensively studied for its health
benefits due to its allicin content. To enhance SAG's drug delivery capabilities, this study aimed to characterize transfersome
formulations containing SAG, investigating their phytochemical activities and the effects of different absolute solvents.
Transfersome formulations, consisting of soy-phospholipid and either Span-60 (T1) or Tween-80 (T2), were prepared using
absolute ethanol (EA) or a chloroform-methanol mixture (CM). Characterization included particle size, polydispersity index
(PDI), and zeta potential. Phytochemical tests assessed antioxidant activity, total phenolic content, and total flavonoid content.
Results showed that T2-CM formulations exhibited the best PDI (0.372 ± 0.022), smallest particle size (T1-CM: 84.333 ±
1.762 nm), and lowest zeta potential (T2-EA: -25.667 ± 0.666 mV). Additionally, T1-CM and T2-CM formulations
demonstrated superior antioxidant, flavonoid, and phenolic content compared to T1-EA and T2-EA. Transfersomes
formulated with organic solvents like absolute ethanol, methanol, and chloroform exhibit promising characteristics and can
effectively protect the antioxidant compounds, flavonoids, and phenols present in SAG extracts. These solvents, known for
their ability to dissolve polar and nonpolar compounds, facilitate the formation of stable, well-characterized transfersomes.
These findings suggest that transfersomes prepared with chloroform-methanol mixtures are more promising for SAG delivery.
Key words: Organic solvents, Phytochemicals, Single aged garlic, Transfersomes

INTRODUCTION
Garlic (Allium sativum L.) is one type of plant that
has been widely used both in the field of food and
health. Garlic bulbs contain allicin and sulfur amino
acid alliin. Studies have shown that garlic has various
medical effects, such as antibacterial, antifungal,
antihypertensive, and anticancer properties. Garlic
also has antioxidant properties (Capasso, 2013; Lee
& Gao, 2012; Sanjay et al., 2018). There are several
varieties of garlic, one of which is single garlic (A.
sativum var. Solo Garlic). Single garlic consists of only
one clove and has the highest antioxidant activity
among the three varieties of garlic. The IC50 value of
the garlic extract using with 70% ethanol is 10.61
µg/mL, lower than that of local garlic Ciwidey (13.61
µg/mL) and imported garlic (11.32 µg/mL), indicating
its stronger antioxidant activity. The ethanol extract
was in the form of a thick extract (Ilmawati et al., 2017;
Qadariah et al., 2020).

One form of single garlic consumption is single
aged garlic (SAG). SAG is a fermented product of

Single garlic that is brewed at 65-80°C with a humidity
of 70-80% of room temperature for 21 days. SAG has
a black color and light mass due to its reduced
moisture content, and has an aroma and taste that is
not too pungent like garlic (Lu et al., 2017). SAG
antioxidants have stronger activity compared to garlic
with Trolox Equivalent Antioxidant (TEAC) values of
13.3 ± 0.5 and 59.2 ± 0.8 mol/g wet, respectively
(Choi et al., 2014). SAG, containing S-allycysteine,
exhibits twice the antioxidant and antibacterial potency
of regular garlic. Its potent antioxidant activity,
attributed to elevated flavonoid and polyphenol levels
from the fermentation process, effectively scavenges
free radicals (Lawson & Hunsaker, 2018; Tran et al.,
2018). The susceptibility of these bioactive compounds
to oxidation and degradation diminishes their
therapeutic efficacy. The implementation of modified

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drug and dosage delivery systems for SAG is
necessary. Modification of preparations in the form of
delivery systems using vesicle systems such as
liposomes, transfersomes and ethosomes has been
reported to increase the ability of drugs to penetrate
the skin (Chaurasiya et al., 2019; Sharma et al.,
2014).
Given the dual nature of SAG's
phytochemicals, the versatile transfersome delivery
system is ideal for optimizing the penetration,
bioavailability, and stability of active compounds,
paving the way for innovative skin care products
(Mitchell et al., 2021). Transfersomes are ultraflexible that can easily penetrate the skin and
overcome the stratum corneum barrier by disrupting
its lipid structure. Transfersomes can deform and
pass through pores that are 5-10 times smaller than
their diameter, and have high entrapment efficiency
for lipophilic drugs, reaching up to 90%.
Transfersomes are composed of hydrophobic and
hydrophilic components, making them suitable for
drugs with low deformability (Chaurasiya et al.,
2019; Sachan et al., 2013).
Transfersomes consist of phospholipids such as
phosphatidylcholine as vesicle-forming components,
surfactants as edge activators to increase flexibility,
absolute ethanol, methanol, and chloroform as
solvents, and buffer solutions as hydrating media
(Omar et al., 2019). Choosing the right solvent greatly
affects how stable and effective the drug delivery
system is when making transfersomes from SAG
extract. Absolute ethanol is used due to its strong
polarity, which enables it to effectively dissolve various
bioactive compounds present in the SAG extract, such
as flavonoids, phenolic compounds, and alkaloids
(Chang et al., 2018; Okonogi et al., 2021). These
bioactive molecules contribute to the therapeutic
effects of the extract, and their proper dissolution is
crucial for ensuring uniform distribution within the
transfersomes.
Additionally,
absolute
ethanol
enhances the fluidity of the lipid bilayer, improving the
vesicles' ability to penetrate biological membranes
(Sharma et al., 2014; Singh & Gaikwad, 2021).
Meanwhile, a combination of methanol and
chloroform is utilized to dissolve specific classes of
compounds that are not efficiently solubilized by
ethanol alone. Methanol, being a polar solvent, is
particularly effective in extracting hydrophilic
substances such as certain flavonoids and phenolic
acids (Jang et al., 2017). In contrast, chloroform, a
non-polar solvent, facilitates the dissolution of
lipophilic compounds, including phospholipids and
certain terpenoids. This biphasic solvent system
ensures comprehensive extraction of both hydrophilic
and lipophilic constituents, optimizing the formulation
and stability of transfersomes (Krishna, 2023; Rasheed
et al., 2022). Absolute ethanol is more polar than the
mixture of methanol and chloroform because the nonpolar chloroform reduces the overall polarity of the
mixture (Apsara et al., 2020; Rasheed et al., 2022). In

the thin-film hydration method for making
transfersomes, lipids and surfactants must be
dissolved in organic solvents until homogeneous. The
selected solvent must be soluble in both the lipid
component and water (Apsara et al., 2020). The
selection of organic solvents in the preparation of
transfersomes is important. The composition of lecithin
as phospholipids and surfactants is a variable that can
affect the optimization of the transfersome formula
(Khan et al., 2022; Surini & Djajadisastra, 2018).
Some surfactants that can be used for transfersome
formulations include single-chain surfactants that can
disrupt the lipid bilayer and increase transfersome
deformability. Examples include sodium cholate,
sodium deoxycholate, span-60, span-65, span-80,
tween-20, tween-60, tween-80, and dipotassium
glycyrrizinate (Khan et al., 2022; Sachan et al., n.d.).
Span-60 and tween-80 have high skin penetration
and smaller particle size, which can increase flexibility
in the lipid bilayer membrane of lecithin vesicles,
allowing transfersomes to pass through pores smaller
than their size spontaneously (Leonyza & Surini, 2019).
This study was conducted to characterize transfersome
formulas and the phytochemical activities of SAG with
different absolute organic ethanol solvents and
chloroform methanol mixtures. Characterization
included particle size, polydispersity index, and zeta
potential. Phytochemical testing included antioxidant
activity using the DPPH method, total phenol content,
and total flavonoid content.
EXPERIMENTAL SECTION
Materials
This research utilized the following materials: single
garlic sourced from Sarangan Village, Magetan
District, East Java, Indonesia, absolute ethanol (Merck,
Germany), Methanol (Merck, Germany), chloroform
(Merck, Germany), phospholipid (Sigma Aldrich,
Germany), Tween-80 (Sigma Aldrich, Germany),
Span-60 (Sigma Aldrich, Germany), 2,2-Diphenyl-1picrylhydrazyl (Sigma Aldrich, Germany), NaNO3
(Merck, Germany), AlCl3 (Smart Lab, Indonesia),
NaOH (Merck, Germany), Folin-Ciocalteu reagent
(Merck, Germany), Na2CO3 (Merck, Germany),
Quercetin (Sigma Aldrich), and Gallic acid (Sigma
Aldrich, Germany).
The tools employed in this research included the
Particle Size Analyzer (HORIBA SZ-100, Japan),
Transmission Electron Microscope (TEM) (Tecnai 200
kV
D2360
SuperTwin,
Japan),
UV-VIS
spectrophotometer (Libra S11/12 Visible & UV
Spectrophotometers,
UK),
magnetic
stirrer
(Thermolyne Cimarec® 2, USA), sonicator (IWAKI
Ultrasonic Cleaner, Japan), analytical balance
(OHAUS), Microwave Assisted Extraction (MAE) device
(Microwave Reaction System SOLV Multiwave PRO,
Anton Paar, Austria), rotary vacuum evaporator
(Heidolph Rotary Evaporator Hei-VAP Expert,
Germany), and waterbath (STUART SBS40, UK).

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Sri Rahayu Lestari, et al.

Extraction of Single Aged Garlic (SAG)
SAG is made by cleaning the dirt from the garlic
skin and then fermenting it at 75°C for 21 days. The
finished SAG is characterized by a change in color
from the original white to a blackish-brown. SAG is
inserted into the vessel of the Microwave Assisted
Extraction (MAE) device (Microwave Reaction System
SOLV Multiwave PRO, Anton Paar, Austria). SAG is
peeled, crushed, and then mixed with absolute ethanol
(Merck, Germany) in a 1:10 ratio in the MAE vessel.
The vessel is closed, arranged in the MAE, and
operated at a holding temperature of 50°C for 10
minutes with a power of 1500 W. The solvent is
evaporated using a rotary vacuum evaporator
(Heidolph Rotary Evaporator Hei-VAP Expert,
Germany) set at a speed of 50 rpm and a temperature
of 37°C. The evaporation results are heated in the
oven at 40°C to remove excess solvents and obtain
pure SAG extract.
Transfesome Formulation as Drug Delivery for SAG
active Compounds
Transfersome preparation was carried out using a
modified thin-film hydration method (Omar et al.,
2019). The phospholipid component (Sigma Aldrich,
Germany) weighed 0.167 g, and edge activator
consisting of surfactants 0.033 g of Tween-80 (Sigma
Aldrich, Germany), and Span-60 (Sigma Aldrich,
Germany) were dissolved in a mixture of organic
solvents chloroform and methanol (CM) (2:1, v:v) and
absolute ethanol (EA) (Merck, Germany). T1 contains
phospholipid and Tween-80, while T2 contains
phospholipid and Span-60. The mixture was heated in
a waterbath (STUART SBS40, UK) at 50 °C, stirred
for 1 hour, and then evaporated. A thin film was
formed after the solvent evaporation stage. The thin
film was hydrated with phosphate-buffered saline
(PBS) containing 0.01 g SAG. The hydration results
were then sonicated (IWAKI, Japan) for 30 minutes,
stirred again for 1 hour, and stored at 4 °C.

Characterization of T-SAG

Particle size characterization was performed to
confirm that T-SAG particles are in the nanoscale
range. The test was conducted at the Integrated
Laboratory & Research Center (ILRC) of the University
of Indonesia. Measurements of particle average (Zaverage), polydispersity index
(PDI), and zeta
potential of T-SAG were carried out using a Particle
Size Analyzer (PSA) (Horiba SZ 100z, Japan), with
three repeated measurements.

2,2-Diphenyl-1-picrylhydrazyl
Activity

(DPPH)

Antioxidant

The DPPH antioxidant test was conducted with
modifications (Lestari et al., 2023). DPPH (Sigma
Aldrich, Germany) was prepared at a concentration of
50 μM in methanol solvent. Transfersome samples
and DPPH solution were mixed in a 1:5 (v/v) ratio and
incubated for 15 minutes at room temperature in dark
conditions. The absorbance of the DPPH mixture and

sample was then measured using a UV-Vis
spectrometer (Biochrom 12S, UK) at a wavelength of
517 nm. The resulting absorbance value was entered
into Equation 1.
%Inhibition:

𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒−𝑆𝑎𝑚𝑝𝑙𝑒 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒
𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒

𝑥 100% (1)

Total Flavonoid Content (TFC)
Each 200 μL transfersome sample was added to
280 μL of dH2O and 60 μL of 5% NaNO3 (Merck,
Germany) and incubated at room temperature in dark
conditions for 5 minutes. After incubation, 60 μL of
10% AlCl3 (Smart Lab, Indonesia) was added in dark
conditions for 6 minutes. The reaction was stopped by
adding 400 μL of 1 M NaOH (Merck, Germany). The
TPC of each transfersome's formula was initially
determined from the standard curve of Quercetin
prepared range from 1 to 50 mg/L solutions of gallic
acid in water. Samples that positively contain
flavonoids will change color to yellow. The results
were then read at 510 nm absorbance on the
spectrophotometer, and the absorbance data results
were compared with quercetin (Sigma Aldrich) as a
standard (Mohammed & Abdullah., 2022). Total
flavonoid levels are expressed in QE (Quercetin
Equivalent), which is the microgram equivalent
amount of quercetin in 1 gram sample (μg QE/g).
Total Phenol Content (TPC)
TPC testing was carried out using the FolinCiocalteu method (Shao et al., 2014). A sample with
a quantity of 400 μL was added to 400 μL of 10%
Folin-Ciocalteu reagent (Merck, Germany) and
incubated at room temperature for 5 minutes. A total
of 300 μL of 75 g/L Na2CO3 (Merck, Germany) was
added at the end of the incubation time and incubated
for 1 hour. Absorbance was observed with a UV-Vis
spectrophotometer at a wavelength of 760 nm, using
gallic acid (Sigma Aldrich, Germany) as a standard.
The TPC of each sample was determined from the
generated standard curve prepared using gallic acid
(GA) ranging from 1 to 50 mg/L solutions of GA in
water. The total concentration of phenol is expressed
in GA, i.e. the microgram equivalent amount of GA in
1 gram sample (μg GAE/g).
RESULTS AND DISCUSSIONS
Characterization of T-SAG
The characterization of Transfersome Single Aged
Garlic (T-SAG) prepared with different surfactants and
solvents revealed distinct physicochemical properties.
The polydispersity index (PDI), z-average, and zeta
potential were assessed to evaluate the size
distribution, particle size, and surface charge of the TSAG, respectively (Table 1). Transfersomes are
phospholipid vesicles employed as carriers for
transdermal drug delivery. Formulations typically
include phospholipids as vesicle-forming agents,
surfactants for flexibility, absolute ethanol, chloroform,
or methanol as solvents, and buffer agents for wetting
(Chaurasiya et al., 2019; Opatha et al., 2020).

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The characterization data presented in Table 1
provide insights into the physicochemical properties of
SAG-Transfersomes
formulated
with
different
surfactants and solvents. The PDI values, all below 0.5,
indicate a relatively uniform size distribution for all the
simulated transfersomes (Chaerunisaa et al., 2023).
The lowest PDI value observed in the T2-EA
formulation suggests a more homogeneous particle
size distribution, which could potentially improve the
stability and bioavailability of the encapsulated SAG.
The z-average measurements reveal that the particle
size of the SAG-loaded transfersomes varied
significantly depending on the surfactant and solvent
combination (Jiang et al., 2018). The T2-EA
formulation, using Span-60 and absolute ethanol,
resulted in the largest particle size. This could be
attributed to the less hydrophilic nature of Span-60,
which might lead to the formation of larger
aggregates or micelles. In contrast, the T1-EA
formulation, using Tween-80 and absolute ethanol,
exhibited the smallest particle size, potentially due
to the better compatibility between Tween-80 and
the phospholipids, resulting in more compact and
smaller transfersomes. An ideal transfersome size is
below 200 nm, as vesicle size is a critical factor
influencing drug encapsulation efficiency during
transfersome manufacturing (Bnyan et al., 2019;
Opatha et al., 2020).
The zeta potential values provide information
about the surface charge of the transfersomes
(Surini & Joshita Djajadisastra, 2018). A more
negative zeta potential can contribute to the
stability of the formulation by minimizing electrostatic

repulsion between particles (Surini & Djajadisastra,
2018). The T2-EA formulation exhibited the lowest
zeta potential, suggesting a potentially higher
stability compared to the other formulations. On the
other hand, the T2-CM formulation, using Span-60
and a chloroform-methanol mixture, displayed the
highest zeta potential, which might indicate a
greater tendency for aggregation. A zeta potential
below -30 mV or above +30 mV is generally
considered stable, preventing particle aggregation
and flocculation (Zhang et al., 2015). Negative
zeta potentials are favorable for stability and
enhance transfersome penetration (Khan et al., 2022;
Nangare et al., 2021).
2,2-Diphenyl-1-picrylhydrazyl (DPPH) Antioxidant
Activity
The results presented in Figure 1 demonstrate the
successful preservation of antioxidant activity in SAG
extract through the transfersome manufacturing
process. The observed differences in antioxidant
activity between transfersomes formulated with
different solvents highlight the impact of the solvent on
the stability and bioactivity of the encapsulated SAG.
Transfersomes prepared with a chloroform-methanol
solvent mixture consistently exhibited higher
antioxidant activity compared to those using absolute
ethanol. This could be attributed to the superior solvent
properties of the chloroform-methanol mixture in
preserving the bioactive compounds of SAG during the
encapsulation process. Chloroform and methanol are
known to be effective solvents for extracting and
solubilizing plant-based compounds, including
antioxidants (Jang et al., 2018).

Table 1. SAG-Transfersome characterization data results
No
Formulation
PDI
Z-Average (nm)
Zeta Potensial (mV)
1
T1-CM
0.399 ± 0.038
84.333 ± 1.762
-14.133 ± 0.839
2
T2-CM
0.372 ± 0.022
174.167 ± 3.166
-16.200 ± 0.361
3
T1-EA
0.382 ± 0.044
96.150 ± 3.889
-12.000 ± 1.058
4
T2-EA
0.409 ± 0.023
366.950 ± 48.578
-25.667 ± 0.666
Note: CM: chloroform methanol 2:1. EA: absolute ethanol. T1: formula phospolipid and tween-80; T2:
formula phospolipid and span-60. PDI : polydispersity index

Figure 1. Antioxidant activity of transfersomes. CM: chloroform methanol. EA: absolute ethanol. T1:
formula phospolipid (SPL) and tween-80 (TWE); T2: formula phospolipid (SPL) and span-60 (SPN).
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Sri Rahayu Lestari, et al.

The T1-EA formulation, using Tween-80 and
absolute ethanol, displayed the lowest antioxidant
activity. This might be due to the less efficient
encapsulation of SAG in absolute ethanol, leading to
a loss of bioactive compounds during the formulation
process. Additionally, the less polar nature of absolute
ethanol compared to the chloroform-methanol
mixture might not be as conducive to maintaining the
stability of the encapsulated antioxidants (Januarti et
al., 2019; Li et al., 2021). Interestingly, when
comparing ethanol-based transfersomes, T2-CM
(using Span-60 and chloroform-methanol) exhibited
slightly higher antioxidant activity than T2-EA (using
Span-60 and absolute ethanol). This suggests that the
choice of surfactant also plays a role in influencing the
antioxidant activity of the transfersomes. The specific
mechanisms underlying these differences require
further investigation.
Phenolic and flavonoid compounds possess
antioxidant properties, acting as free radical
scavengers. Their chemical properties, including
hydrogen atom donation, metal chelation, and
biological activity, contribute to their antioxidant
effects (Elosta et al., 2017; Mohammed & Abdullah,
2022). Previous studies reported no significant impact
of encapsulation on resveratrol's antioxidant activity in
transfersomes (Wu et al., 2019). Additionally,
tetrahydrocurcumin's antioxidant activity was not
hindered by its encapsulation or incorporation into
vesicular cream (Kanshide et al., 2022). Aged garlic
extract itself exhibits antioxidant activity in various
solvents, including distilled water, ethanol, and
chloroform (Jang et al., 2018). SAG has been shown
to contain higher levels of phenols, flavonoids, and
sulfur compounds (e.g., S-ally-(L)-cysteine, disulfide)
compared to fresh garlic. The abundance of phenolic
and flavonoid compounds correlates positively with
DPPH radical scavenging activity, attributed to their
ability to donate hydrogen and electrons from the
hydroxyl group (Eun et al., 2014; Jang et al., 2018;
Lu et al., 2017).
Total Flavonoid Content (TFC)
The data presented in Table 2 reveal variations in
total flavonoid content (TFC) among the different black
garlic Transfersome formulations. T2 formulations,
utilizing Span-60 as the surfactant, consistently
exhibited higher TFC values compared to T1
formulations, employing Tween-80. This suggests that

Span-60 might be more effective in encapsulating and
protecting flavonoids during the transfersome
formation process. The solvent type also influenced
TFC. Both T1 and T2 formulations using chloroformmethanol (CM) solvent demonstrated slightly higher
TFC values than those using absolute ethanol (EA).
This could be attributed to the superior solvent
properties of chloroform-methanol in extracting and
solubilizing flavonoids from the single aged garlic
extract.
Flavonoids are the largest group of phenolic
compounds that have properties as antioxidants
(Hanin & Pratiwi, 2017; Safe et al., 2021). As in the
total phenolic content test, the increased antioxidant
activity of SAG is due to an increase in the bioactivity
of flavonoid compounds, polyphenols, and the
formation of SAC compounds (Sasmitaloka et al.,
2022; Sembiring & Iskandar, 2019). While
organosulfur compounds, such as S-allyl cysteine
(SAC) and diallyl disulfide, are indeed the
characteristic active compounds in garlic, our study
also considers flavonoid and phenolic compounds as
important bioactive components in single-aged garlic
(SAG) (Dewi, 2018). Notably, black garlic, including
SAG, contains polyphenolic compounds such as
quercetin, kaempferol, catechin, and gallic acid,
which contribute significantly to its antioxidant
properties (Ryu & Kang, 2017). These compounds
enhance the bioactivity of SAG and influence its
pharmacological potential. Since flavonoids and
phenolic compounds exhibit strong antioxidant
activity, they are crucial parameters for evaluating the
effectiveness of transfersomes in delivering these
bioactive components (Nguyen et al., 2021; Ryu &
Kang, 2017). Total flavonoid content is more identified
in transfersomes with methanol and chloroform
solvents than absolute ethanol solvents. Solvents like
methanol and chloroform are crucial for solubilizing
lipids and forming stable transfersome structures
(Jiang et al., 2018; Nayak & Tippavajhala, 2021).
The interaction between these solvents and lipids
significantly influences the size, morphology, and
cargo capacity of the vesicles, as well as the total
encapsulated flavonoids. Soy lecithin, a common
phospholipid used in lipid-based vesicular drug
delivery systems, typically constitutes 1-4% w/w of
the formulation (Chaurasiya et al., 2019; Leonyza &
Surini, 2019). Soy lecithin is readily available and
relatively inexpensive.

Table 2. Total flavonoid content of transfersome SAG extract
Total Flavonoid Content (μg QE/g)
No
Formulation
1
T1-EA
38.38 ± 0.68
2
T2-EA
62.55 ± 0.34
3
T1-CM
40.78 ± 0.48
4
T2-CM
66.61 ± 0.18
Note: CM: chloroform methanol 2:1. EA: absolute ethanol. T1: formula phospolipid and tween-80;
T2: formula phospolipid and span-60. PDI : polydispersity index
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Table 3. Total phenolic content of transfersome SAG extract
No

Formulation

Total Phenolic Content (μg GAE/g)

T1-EA
50.18 ± 1.73
1
2
T2-EA
43.83 ± 1.36
3
T1-CM
51.80 ± 2.76
4
T2-CM
49.39 ± 0.53
Note: CM: chloroform methanol 2:1. EA: absolute ethanol. T1: formula phospolipid and tween-80;
T2: formula phospolipid and span-60. PDI : polydispersity index
Tween-80 and Span-60, used as penetration
enhancers, are incorporated at 5-10%, while ethanol
is used at 20-40%. High ethanol concentrations
can tighten the lipid membrane, enhancing
stability and creating a softer structure, which improves
drug distribution in the stratum corneum (Leonyza &
Surini, 2019; Wongrakpanich et al., 2022a).
Additionally, Tween-80 and Span-60 enhance the
flexibility of the lecithin vesicle's lipid bilayer, allowing
transfersomes to spontaneously pass through pores
smaller than their size (Khan et al., 2022; Leonyza &
Surini, 2019).
Total Phenol Content (TPC)
Total
phenolic
content
in
SAG-loaded
transfersomes was determined using gallic acid as a
standard, due to its natural phenolic nature and
stability. Table 3 summarizes the absorbance values
and total phenolic content of the T-SAG transfersomes.
The data obtained from this study revealed significant
variations TPC among different formulations of single
aged garlic extract transfersomes. These variations
were primarily attributed to differences in the types of
solvents and surfactants employed in transfersome
preparation.
Transfersomes formulated with CM solvents
consistently exhibited higher TPC values compared to
those using EA. This suggests that the solvent's polarity
and its ability to dissolve phenolic compounds
significantly influence encapsulation efficiency
(Jahanfar et al., 2021; Vo et al., 2019). The
chloroform-methanol mixture, due to the presence of
chloroform as a non-polar component, effectively
dissolves non-polar phenolic compounds in single
aged garlic extract, facilitating their incorporation into
the transfersome structure. At the same time, the
presence of methanol increases the solvent system’s
overall polarity, improving its ability to dissolve
amphiphilic components such as phospholipids and
surfactants. This dual nature of the solvent system
enhances the efficiency of transfersome formation by
optimizing the solubility of both hydrophobic and
hydrophilic compounds (Fitrya et al., 2020; Lu et al.,
2017; Singh & Gaikwad, 2021). Solvents like
methanol and chloroform are crucial for solubilizing
lipids and forming stable transfersome structures. The
interaction between these solvents and lipids
significantly influences the vesicles’s size, morphology,
and cargo capacity and the total encapsulated
phenols (Natsheh & Touitou, 2020). Soy lecithin, a

commonly used phospholipid in lipid-based vesicular
drug delivery systems, typically constitutes 1- 4% w/w
of the formulation (Chaurasiya et al., 2019). It is
readily available and relatively inexpensive. Methanol
also aids in phospholipid hydration, a key component
of the transfersome membrane (Abdel-Gawad et al.,
2018; Mohammed & Abdullah, 2022). The interaction
between methanol, phospholipids, and SAG
compounds during the preparation process influences
vesicle size, distribution, and encapsulation efficiency.
In addition to methanol, chloroform is another solvent
used in transfersome preparation. The combination of
chloroform and methanol is particularly effective for
dissolving Tween-80 or Span-60 surfactants and
efficiently absorbing SAG extract. Chloroform's high
solubility in alcohol and ether makes it a suitable
solvent for use with methanol (Natsheh & Touitou,
2020).
Surfactants also played a crucial role in TPC.
Tween-80, being more hydrophilic than Span-60,
tended to produce formulations with slightly higher
TPC values. This indicates Tween-80's superior ability
to stabilize emulsions and protect phenolic
compounds
from
degradation
during
the
transfersome manufacturing process. The interaction
between solvents, surfactants, and phenolic
compounds is a complex factor influencing TPC in
transfersomes (Jiang et al., 2018; Leonyza & Surini,
2019). Solvents affect the solubility of phenolic
compounds, while surfactants influence micelle
formation and emulsion stability (Elosta et al., 2017;
Lu et al., 2017). The combination of chloroformmethanol solvent and Tween-80 surfactant proved
particularly effective in maintaining TPC, likely due to
its ability to form a stable transfersome structure and
efficiently encapsulate phenolic compounds (Khan et
al., 2022). These findings have significant implications
for the development of transfersome-based
formulations for bioactive compound delivery. The
careful selection of solvents and surfactants can
substantially impact the encapsulation efficiency and
stability of active compounds within the formulation.
The combination of methanol and chloroform
enhances the solvent's polarity, increasing its ability to
dissolve phospholipids and surfactants like Tween-80
or Span-60, resulting in a homogeneous mixture
within the transfersome formulation (Islam et al.,
2021; Wongrakpanich et al., 2022b). Transfersomes
formulated with organic solvents like absolute ethanol,

296

Effect of Organic Solvents in the Preparation

Sri Rahayu Lestari, et al.

methanol, and chloroform exhibit promising
characteristics and can effectively protect the
antioxidant compounds, flavonoids, and phenols
present in SAG extracts. These solvents, known for
their ability to dissolve polar and nonpolar
compounds, facilitate the formation of stable, wellcharacterized transfersomes. The combination of
these solvents can enhance the encapsulation
efficiency of bioactive compounds, protecting them
from degradation and ensuring their controlled
release.
The
resulting
transfersomes
often
demonstrate desirable properties, such as small
particle size, uniform distribution, and high stability,
making them suitable for various applications,
including
drug
delivery
and
nutraceutical
formulations.
CONCLUSIONS
Transfersomes composed of phospholipids and
Tween-80 were found to be the most effective
formulation based on solvent type. Among the solvents
tested, ethanol was superior in preserving antioxidant
activity compared to chloroform and methanol.
Furthermore, the SAG transfersomes formulated using
a chloroform-methanol solvent mixture exhibited
superior phytochemical characteristics and a higher
content of phenolic and flavonoid compounds
compared to those formulated with absolute ethanol.
This suggests that the chloroform-methanol mixture
provided better protection and retention of these
bioactive compounds within the transfersome
structure. To further support these findings, additional
research is necessary to investigate encapsulation
efficiency, drug release, and other in vitro parameters.
ACKNOWLEDGEMENTS
The authors would like to express their gratitude to
the Research and Community Service Institution (LPPM)
of Universitas Negeri Malang for providing funding for
our international collaborative research under contract
number 5.4.1/UN32/KP/2023. Additionally, we
would like to acknowledge the invaluable support of
the SRL Team in facilitating this research.
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