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Asian Journal of Healthy and Science
p-ISSN: 2980-4302
e-ISSN: 2980-4310
Vol. 3 No. 12 December, 2024
Formulation and Characterization of Mucolytic Nanosuspension of Iler Leaf
Extract (
Coleus atropurpureus L. benth.
) With Sodium Alginate as a Suspending
Agent
Akhmad Fadhil Samara1*, Noval2, Mia Audina3, Setia Budi4
Universitas Sari Mulia, Indonesia
Emails: fadhil.smara@gmail.com1, noval@unism.ac.id2,
miaaudina@unism.ac.id3, setiabuddi62@gmail.com4
Abstract
Iter leaves (
Coleus atropurpureus L. Benth.
) are known for their mucolytic properties, which
help to thin phlegm. However, poor absorption and bioavailability of some active
components in herbal extracts, due to their large molecular size and inability to penetrate
lipid membranes, limit their therapeutic potential. This research aimed to develop a
nanosuspension formulation with fine particles to improve dissolution rate and
bioavailability. An experimental method with a Non-Equivalent Control Group Design was
used. Nanosuspensions were formulated using iler leaf extract with sodium alginate as the
suspending agent. Characterization tests included organoleptic evaluation, pH, density,
viscosity, redispersion, particle size distribution, zeta potential, and sedimentation volume.
The results showed that all formulations met the required quality standards. pH ranged
from 6.06 to 6.14 (p < 0.001), density ranged from 1.015 to 1.044 g/ml (p < 0.01), viscosity
ranged from 38.2 to 93.8 cPs (p < 0.001), and redispersion testing confirmed 100%
redispersion without clumping after three treatments. Particle size distribution ranged from
69.2 to 898.8 nm (p < 0.001), zeta potential ranged from -33.15 to -37.38 mV (p = 0.35),
and sedimentation volume ranged from 0.95 to 0.98 ml. This research has implications
implying that nanosuspensions of iler leaves, optimized with varying concentrations of
sodium alginate, offer a promising strategy to improve the bioavailability and therapeutic
efficacy of mucolytic herbal preparations. These results open up opportunities for further
research regarding in vivo evaluation, development of combination therapies, and
production scale-up to enable industrial application and commercialization in the
pharmaceutical market.
Keywords: Iler Leaf Extract, Mucolytic, Nanosuspension, Sodium Alginate.
INTRODUCTION
The iler plant (
Coleus sp.
) is a medicinal plant which is also an ornamental plant that is
quite popular with the people of Indonesia. From various studies that have been conducted,
iler leaves (
Coleus atropurpureus L. benth.
) have properties as mucolytics or cough
medicines that function to thin phlegm. In research conducted by (Herdaningsih &
Kartikasari, 2022), iler leaf extract (
Coleus atropurpureus L. benth
.) at a concentration of 3-
6% has mucolytic activity equivalent to the positive control (acetylcysteine 0.2%). Based on
these results, iler leaf extract can be used as an alternative in the treatment of cough
(Herdaningsih & Kartikasari, 2022). However, some of the active components present in
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349
herbal extracts are unable to pass through lipid membranes because they have a very high
molecular size or poor water solubility, resulting in low absorption and poor bioavailability
(Fadlilah & Gozali, 2022).
One of the important drug delivery systems to be developed today is nanosuspensions.
Nanosuspensions are described as very fine particles, biphasic, solid particles separated in
an aqueous carrier and stabilized by surfactants with reduced particle size, with the aim of
making better dissolution rates and increasing bioavailability. In addition to increasing
bioavailability, nanosuspensions can also increase absorption and biological action because
nanosuspensions have good dissolution rates and drug saturation (Fadlilah & Gozali, 2022).
The diameter of the suspended particles is less than 1 µm, which is 0.1 nm-1000 nm.
One thing that needs to be considered in developing a suspension dosage form is the
suspending agent. Of the many types of suspending agents used for nanosuspension
technology, sodium alginate is one of the anionic polymers that has been widely studied in
the controlled release of pharmaceutical preparations with other polymers due to its low cost
(Frent et al., 2022).
Despite the growing body of research on nanosuspensions, there remains a theoretical
gap in understanding how variations in suspending agents, particularly sodium alginate,
influence the physicochemical properties and stability of herbal-based nanosuspensions.
Additionally, the mechanisms by which nanosuspensions enhance the bioavailability and
therapeutic efficacy of high-molecular-weight herbal compounds require further exploration.
Addressing these gaps is crucial to optimize the formulation and ensure effective drug
delivery.
In practical terms, the development of nanosuspensions faces challenges in achieving
consistent particle size reduction, ensuring stability during storage, and maintaining the
desired physicochemical properties. Furthermore, scaling up nanosuspension production for
commercial applications while retaining quality and efficacy remains a significant obstacle.
These practical challenges underline the importance of systematic formulation studies and
characterization to facilitate the successful implementation of nanosuspension technology in
herbal medicine.
Based on the above considerations, this research aims to formulate and characterize
mucolytic nanosuspensions from iler leaf extract (
Coleus atropurpureus L. Benth.
) using
sodium alginate as a suspending agent. The goal is to develop an effective drug delivery
system for phlegm thinners or mucolytics, addressing both theoretical and practical
challenges in the field. So that the benefits in this research are to make a significant
contribution in the pharmaceutical field, especially in the development of innovative and
effective nanosuspension-based drug preparations. This research is expected to improve the
efficacy of mucolytic drug delivery, extend the stability of the preparation, and optimize
patient safety and comfort. In addition, another benefit is to open up opportunities for wider
application of natural materials, such as iler leaf extract, as an alternative in modern
pharmaceutical technology-based medicine. The results of this research are also expected to
be a scientific reference for further research and have a positive impact on the
pharmaceutical industry.
RESEARCH METHOD
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The research method used in this research is the Quasy Experimental Design method
with the Non Equivalent Control Group Design. The data collection instruments used in this
research are a pH meter which is an instrument used to measure the pH of the preparation,
a Stormer Viscometer is an instrument used to measure the viscosity or viscosity of the
preparation, a Picnometer is an instrument used to measure density, then there are
glassware such as test tubes and 10ml measuring cups used to conduct redispersion tests and
sedimentation volume, and the last is the Particle Size Analyzer (PSA) Malvern Zetasizer Pro
(ZSU 300) is an instrument used to measure particle size distribution and zeta potential.
The types of qualitative data used in this research are organoleptic tests and
redispersion tests. While the quantitative data used in this research are pH content test,
density test, viscosity test, particle size distribution test, zeta potential, and sedimentation
volume.
The tools used in this research are glassware (Pyrex, Herma), oven, blender, rotary
evaporator (Dlab), analytical balance (ACIS AD-300i), Stormer Viscometer, Picnometer, pH
meter, Particle Size Analyzer (PSA) Malvern Zetasizer Pro (ZSU 300), Overhead Stirrer, Hand
Homogenizer, and Hotplate Stirrer (Thermo scientific). The materials used in this research
are thick extract of iler leaf (Coleus atropurpureus L. benth.), phosphate-buffered saline
solution pH 6, 70% ethanol, aspartame, sodium alginate, propylene glycol, propyl paraben,
sodium thiosulfate, sodium chloride, distilled water.
Table. 1 Formulation of the preparation
Material
Formulation (%)
Material Function
F1
F2
F3
Iler Leaf Extract
6
6
6
Active Substance
Sodium Alginate
1
3
5
Stabilizer
Aspartame
1
1
1
Sweetener
Propylene glycol
15
15
15
Solvent
NaCl
0,05
0,05
0,05
Preservatives
Propyl Paraben
0,02
0,02
0,02
Preservatives
Sodium Thiosulfate
0,02
0,02
0,02
Thickener
Phosphate diluent pH6
0,8
0,8
0,8
pH regulator
Aquadest
Ad 100 ml
Ad 100 ml
Ad 100 ml
Solvent
a. Preparation of Iler Leaf Extract (
Coleus atropurpureus L. benth.
)
Collect 2 kg of iler leaves, then wet sorted by washing using running water until
clean. The clean iler leaves are then dried using an oven. After drying, clean again from
impurities that are still attached. The dried simplisia is then blended until it becomes
powdered simplisia, then do the maceration process by putting all the powdered simplisia
into a closed container and immersed in 300ml of 70% ethanol, then macerated for 3 days,
after which every 24 hours the solvent is replaced by a new solvent. The filtrate was
separated from the residue, then the residue was macerated again in 150 ml of solvent
using the same method. This process is always carried out until the color of the resulting
filtrate is constant. The filtrate is collected and evaporated using a rotary evaporator until
it becomes a thick extract. (Utami et al., 2020).
b. Nanosuspension Formulation of Iler Leaf Extract (
Coleus atropurpureus L. benth.
)
Weigh all ingredients, dissolve sodium alginate powder with water as much as 7
times its weight in a mortar and allowed to expand. Tiler leaf extract was added little by
little with the addition of water and propylene glycol, stirred until homogeneous. Sodium
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chloride and sodium thiosulfate were dissolved in water, then mixed into sodium alginate
and iler leaf extract and stirred until homogeneous. Propyl paraben is dissolved in
propylene glycol, mixed into sodium alginate which has been mixed with iler leaf extract,
water, sodium chloride and sodium thiosulfate and stirred until homogeneous. Aspartame
is dissolved into enough water by heating until homogeneous, after which it is mixed into
the sodium alginate mixture of iler leaf extract and stirred until homogeneous. The
remaining propylene glycol and phosphate dapar solution pH 6 were added to the
suspension preparation that had been mixed until homogeneous, (Muthmainnah, 2020).
Add water up to 100%, then dispersed with a magnetic stirrer at 1000 rpm for 20 minutes,
stirred with a homogenizer at 15,000 rpm for 10 minutes, after which it was put into a
container. (Pınar et al., 2022). Then the evaluation and characterization of
nanosuspension of iler leaf extract (
Coleus atropurpureus L. benth.
) (Dzakwan, 2020).
c. Organoleptic Test
Organoleptic testing aims to assess and identify iler leaf extract nanosuspension
preparations. Identification of iler leaf extract nanosuspension includes odor, color, and
taste (Budiati et al., 2023).
d. pH Level Test
pH testing is carried out using a pH Meter, which aims to determine the amount of acidity
level in a preparation, whether it is in accordance with the provisions or not in accordance
with the provisions (Wijaya & Lina, 2021)
e. Density Test
The density test was conducted using a dry and clean empty 50 ml piconometer. (a).
Then add distilled water into the pycnometer and measure the weight. (b)
Nanosuspension of extract combination was put into the pycnometer and measured the
weight. (c) The density of the extract formulation nanosuspension is determined using the
following formula (Wijaya & Lina, 2021).
𝜌𝑐 − 𝑎
𝑏 − 𝑎 𝑥 𝜌
f. Viscosity Test
The viscosity test was carried out using a Stormer viscometer at a rotation speed of 30
rpm spindle no.1.
g. Redispersion Test
The redispersion test is done manually with care, the bottle containing the formulation is
shaken 10-13 times (Budiati et al., 2023).
h. Particle Size Distribution
Each formulation was evaluated by optical microscope analysis using a 100x (10x10)
ocular lens equipped with a camera. Particle size determination was done by measuring
100 particles from each formulation and categorizing the particle size (Muthmainnah,
2020).
i. Zeta Potential
The zeta potential test of nanoparticles was carried out using a PSA (particle size analyzer)
tool at 25°C. Zeta potential is a charge parameter between nanoparticles. Higher zeta
potential values prevent aggregation (formation of larger colloids from smaller colloids).
The requirement of zeta potential is ± 30 mV (Ambarwati & Rustiani, 2022).
j. Sedimentation Volume Test
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RESULTS AND DISCUSSION
Organoleptical Test
Table 2. Organoleptical Test Results
Formulation
Organoleptical Test
Smell
Color
Taste
1
Distinctive of iler leaf extract
Chocolate
Sweet
2
Distinctive of iler leaf extract
Chocolate
Sweet
3
Distinctive of iler leaf extract
Chocolate
Sweet
From the organoleptical test results above, the four formulas show the same results in
terms of smell, color, and taste, namely having a distinctive smell of iler leaf extract (
Coleus
atropurpureus L. benth
.), brown color, and sweet taste.
pH test
Figure 1. pH test graph
Table 3. pH Test Results
Formulation
Mean±SD
Specifications
Description
I
6,06±0,15
The optimum suspension pH is 5-6
Meet
II
6,13±0,01
Meet
III
6,14±0
Meet
p-Value
0,001a
The pH test results obtained in formulation I amounted to 6.06, formulation II
amounted to 6.13, and formulation III amounted to 6.14. In the results tested, the highest pH
value was in formulation III of 6.14 and the lowest pH value in formulation I of 6.06. The
above data were analyzed using the One-Way ANOVA test obtained a significance value of
0.001a (p <0.05).
Density Test
Table 4. Density Test Results
Formulation
Mean (g/ml) ±SD
Specifications
Description
I
6,06±0,15
The optimum suspension pH is 5-6
Meet
I
1,015±0,11
The specific gravity of the preparation
with water carrier should be more than
1.00 g/mL
Meet
II
1,025±6,03
Meet
p-Value
0,01a
6.06
6.13 6.14
6
6.05
6.1
6.15
Guarantee 1 Guarantee 2 Guarantee 3
pH Test
Ph Tests
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Figure 2. Density Test Chart
The density test results obtained in formulation I amounted to 1.015 g/ml, formulation
II amounted to 1.025 g/ml, and formulation III amounted to 1.044 g/ml. In the results tested,
the highest density test value was found in formulation III at 1.044g/ml and the lowest pH
value in formulation I at 1.015g/ml. The above data was obtained by analyzing the One-Way
ANOVA test with a significance value of 0.10a (p>0.05).
Viscosity Test
Table 5. Viscosity Test Results
Formulation
Average
(cps)±SD
Specifications
Description
I
6,06±0,15
The optimum suspension pH is 5-6
Meet
I
38,2±1,44
The viscosity value of the suspension according
to SNI is 37cP-396 cP.
Meet
II
63,0±1
Meet
p-Value
0,01a
Figure 3. Viscosity Test Chart
The viscosity test results obtained in formulation I amounted to 38.2 cps, formulation
II amounted to 63.0cps, and formulation III amounted to 93.8cps. In the results tested, the
highest viscosity value was in formulation III of 93.8cPs and the lowest pH value in
formulation I of 38.2cPs. The above data was obtained by analyzing the One-Way ANOVA test
with a significance value of 0.001a (p <0.05).
1.015
1.025
1.044
1
1.01
1.02
1.03
1.04
1.05
Formulation 1 Formulation 2 Formulation 3
Density Test (g/cm3)
Density Test
38.2
63
93.8
0
20
40
60
80
100
Formulation 1 Formulation 2 Formulation 3
Viscosity Test (Cps)
Viscosity Test
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Redispersion Test
Table 6. Redispersion Test
Formulation
Number of Treatments
%Redispersion
Caking
I
6,06±0,15
The optimum suspension pH is 5-6
Meet
I
3 times
100%
No
caking
II
3 times
100%
No
caking
The redispersion test results obtained with the number of revolutions to redisperse are
1x rotation (180º), the redispersion results of all preparations in sequence starting from
Formulations I, II, and III are obtained 100% and there is no caking in the preparation when
cornered.
Particle Size Distribution
Table 7. Particle size distribution results
Formulation
Average
(nm)±SD
Specifications
Description
I
69,2±10,4
Nanosuspensions are colloidal dispersion
systems containing drugs with particle sizes of
10-1000 nm.
Meet
II
146,9±21,7
Meet
III
898,8±137,0
Meet
p-Value
0,001a
Figure 4. Particle size distribution graph
The results of the particle size distribution test obtained data on formulation I of 69.2nm,
on formulation II of 146.9nm, and on formulation III of 898.8nm. In the results tested, the
highest particle size distribution value was in formulation III of 898.8nm and the lowest
particle size distribution value in formulation I of 69.2nm. Based on the results that have been
tested, the three formulations all meet the requirements of the Nanosuspension preparation.
The above data was obtained by analyzing the One-Way ANOVA test with a significance value
of 0.001a (p <0.05).
Zeta Potential
Table 8. Zeta Potential Results
Formulation
Average
(-mV)±SD
Specifications
Description
I
-33,15±3,5
Nanoparticles with zeta potential values
smaller than -30 mV and greater than +30
mV have higher stability.
Meet
II
-37,38±3,5
Meet
III
-36,59±3,5
Meet
p-Value
0,35a
69.2 146.9
898.8
0
200
400
600
800
1000
Formulation 1 Formulation 2 Formulation 3
Particle Size Distribution (nm)
Particle Size Distribution
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Figure 5. Zeta potential graph
The zeta potential test results obtained data on formulation I amounted to -28.53mV,
on formulation II -37.38mV, and on formulation III -36.59mV. In the results tested, the highest
potential zeta value is in formulation II of -37.38mV and the lowest potential zeta value in
formulation I of -33.15mV. Based on the results that have been tested in a row into the
requirements but almost meet the requirements, and in formulation II and formulation III the
results obtained are in accordance with the existing requirements. The above data was
obtained by analyzing the One-Way ANOVA test with a significance value of 0.35a (p>0.05).
Sedimentation Volume Test
Table 9. Sedimentation Volume Test Results
Formulation
Mean±SD
Specifications
Description
I
0,95±5,77
A good suspension sedimentation volume test
has a price < 1 or > 1.
Meet
II
0,97±5,77
Meet
III
0,98±5,77
Meet
p-Value
0,033a
Figure 6. Sedimentation Test Chart
Discussion
This research begins with the extraction process of iler leaf (
Coleus atropurpureus L.
benth.
) using the maceration method which is carried out for 3 days by soaking iler leaf
powder (
Coleus atropurpureus L. benth
.). Then proceed with the formulation of
Nanosuspension of iler leaf extract (Coleus atropurpureus L. benth.) using sodium alginate
as a suspending agent which varies in different concentrations in each formula. The variation
33.15
37.38 36.59
30
32
34
36
38
Formulation 1 Formulation 2 Formulation 3
Zeta Potential (-mV)
Zeta Potential
0.95
0.97
0.98
0.93
0.94
0.95
0.96
0.97
0.98
0.99
Formulation 1 Formulation 2 Formulation 3
Sedimentation Test (ml)
Sedimentation Test
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of sodium alginate concentration in formula I is 1g, formulation II is 3g, and formulation III is
5g.
Organoleptical Test
The results of testing the preparation of Nanosuspension of Iler Leaf Extract (
Coleus
atropurpureus L. benth.
) in the three formulations obtained brown results and a distinctive
odor of Iler leaves (
Coleus atropurpureus L. benth.
) in the preparation which is the result of
Iler leaf extract (
Coleus atropurpureus L. benth.
), while the sweet taste in the preparation is
obtained from the sweetener used in the formulation, namely aspartame
.
From the results of the research that has been obtained, it shows that the three
formulations are in accordance with the organoleptical provisions despite the different
concentrations of sodium alginate used, this is because the difference in sodium alginate
concentration from low to high does not significantly affect the color, odor, and taste of the
Nanosuspension preparation of Iler Leaf Extract (
Coleus atropurpureus L. benth.
).
pH Level Test
In general, sodium alginate has a pH of 9-10, and pH values greater than 7 indicate
alkaline properties (Putriyana et al., 2018). The pH test that has been carried out obtained
suitable results at pH 6, this is due to the influence of the addition of phosphate dapar pH 6
in the formulation of making Nanosuspension of Iler Leaf Extract (
Coleus atropurpureus L.
benth
.). However, in the preparations that have been tested there is a slight pH difference
that is not significant but there is still a difference, and it can be seen that the higher the
concentration of sodium alginate, the higher the pH value obtained, this result is in
accordance with the statement that says that the pH properties of sodium alginate are
alkaline (Abka-Khajouei et al., 2022)..
Density Test
Based on the results of density measurements, the average result obtained in
formulation I was 1.015g/ml, formulation II was 1.025g/ml, and in formulation III was
1.044g/ml. Judging from the results obtained, it meets the requirements, which say that the
density or specific gravity of a preparation with a water carrier must be> 1.00g / ml, this is
because water has a specific gravity of 1.00g / ml (Ulfah & Slamet, 2020).
Viscosity Test
Based on the results of viscosity testing that has been carried out, the results of
formulation I with an average of 38.2cPs, formulation II of 63.0cPs, and formulation III of
93.8cPs were obtained. Therefore, the average results obtained by each formulation tested
ranged from 38.2cPs-93.8cPs, and these results of each formulation tested met the range of
viscosity requirements of the Mucolytic Nanosuspension preparation of iler leaf extract
(
Coleus atropurpureus L. benth
.). These results are in accordance with the theory that the
higher the concentration of sodium alginate used, the higher the viscosity of the preparation
(Jayanudin et al., 2014).
Redispersion Test
In this test, the redispersion test was carried out by rotating the preparation 1x (180º),
the results of this test were that each consecutive formulation from formulation I,
formulation II, and formulation III could be dispersed 100% without any caking at the time
of cornering, so that from the redispersion tests that have been carried out on formulation I,
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Particle Size Distribution
The results that have been obtained are certainly influenced by several factors, starting
from the method of making Mucolytic Nanosuspension preparations of iler leaf extract
(
Coleus atropurpureus L. benth.
) using the Top-Down technique of High Pressure
Homogenization type, which technique consists of three steps in the manufacturing method,
the first step of the active substance is dispersed in a stabilizer solution to form a prasuspensi,
then the prasuspensi that has been formed is homogenized using a high pressure
homogenizer at high pressure premilling, which is finally homogenized at high voltage for 10
to 25 cycles until the expected nanosuspension size is formed (Fadlilah & Gozali, 2022).
Factors that affect the particle size distribution in the Mucolytic Nanosuspension
preparation of iler leaf extract (
Coleus atropurpureus L. benth.
) are in the suspending agent
used, namely sodium alginate. Sodium alginate itself is a substance that has soft viscoelastic
properties, which means that the substance has flowing and elastic properties at the same
time, because of this, sodium alginate particles can easily change shape when getting external
stimuli caused by water and its gel network (Abka-Khajouei et al., 2022).. Based on this, it
can be concluded that viscosity and density weight have a relationship with the neatness of a
preparation, which will certainly have an influence on the particle size distribution of
Mucolytic Nanosuspension preparation of iler leaf extract (
Coleus atropurpureus L. benth
.).
Zeta Potential
Zeta potential is a measure of the potential difference between the bulk liquid in which
a particle is dispersed and the liquid layer containing oppositely charged ions associated with
the nanoparticle surface (Selvamani, 2019). In addition, zeta potential will also provide an
overview of the repulsive forces between particles and cause the greater the zeta potential
so that the dispersion system will be more stable (Noval & Malahayati, 2021).
Based on the results of zeta potential testing that has been done, the results of
formulation I with an average of -33.15mV, formulation II of -37.38mV, and formulation III of
-36.59mV are obtained. Therefore the average results obtained for each formulation tested
ranged from -33.15mV-37.38mV, consecutively the smallest particle size distribution results
were in formulation I with a value of -33.15mV and the largest particle size distribution was
in formulation II with a value of -37.38mV. From these results, each formulation tested has
met the range of requirements for the value of the requirements of a good zeta potential test,
which is at least -30mV (Koca et al., 2022).
Sedimentation Volume Test
Based on the test results that have been carried out, the average sedimentation volume
in formulation I is 0.95, formulation II is 0.97, and in formulation III is 0.98. So judging from
the results that have been obtained in all formulas in a row ranging from 0.95-0.98, the results
have met the existing requirements, namely a good sedimentation volume <1. From the
results that have been obtained, it can be concluded that the viscosity or viscosity of the
preparation will affect the speed of the sedimentation process, the greater the level of
viscosity or viscosity in Nanosuspension, the slower the sedimentation process will be, this
is due to the resistance provided by the suspending agent or suspending material.
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CONCLUSION
This research successfully addressed the problem of formulating and characterizing
mucolytic nanosuspensions from iler leaf extract (Coleus atropurpureus L. Benth.) using
sodium alginate as the suspending agent. The research demonstrated that sodium alginate
concentration significantly influenced key physicochemical properties of the
nanosuspensions, including viscosity, pH, particle size distribution, zeta potential, and
sedimentation volume. All formulations adhered to the quality standards for
nanosuspensions, featuring particle size distributions in the nanometer range and high
stability as indicated by zeta potential measurements. These results underscore the potential
of iler leaf nanosuspensions to improve the bioavailability and therapeutic efficacy of
mucolytic herbal extracts, thereby contributing to the advancement of plant-based
pharmaceutical applications.
The research's contributions establish a solid groundwork for the further development
of nanosuspensions derived from natural materials. Future research should prioritize in vivo
evaluations of bioavailability and pharmacological efficacy, development of combination
formulations with complementary active ingredients, and integration with advanced drug
delivery technologies such as controlled release systems. Additionally, investigations into
long-term stability and scalability for industrial production are essential for enabling the
commercialization of nanosuspension-based herbal therapies.
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Copyright holders:
Akhmad Fadhil Samara, Noval, Mia Audina, Setia Budi (2024)
First publication right:
AJHS - Asian Journal of Health and Science
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