Advances in Health Sciences Research, volume 26

2nd Bakti Tunas Husada-Health Science International Conference (BTH-HSIC 2019)

Evaluation Nano-Phytosome of Myricetin

with Thin Layer Film Hydration-Sonication
Method
Yuli Edy Saputra1, Muhammad Dzakwan2, Nur Aini Dewi3
Fakultas Farmasi, Universitas Setia Budi, Surakarta
E-mail: yulled089@gmail.com
Corresponding author: aini_farmasi2008@yahoo.com

Abstract - Nano-phytosome is a nano technology Drug delivery system of the latest generation
that’s use to improve the bioavailability of active has the advantage to improve the nature of
compound in plants by binding with phospholipids penetration of the skin. Recent research in
which have similar characteristic with cell
nanotechnology have enabled the manufacture of
membranes. In this study, Myricetin is the main
ingredient that we used in nano-phytosome
nano-sized particles used for various biomedical
formulation. Myrisetin is a natural flavonoid applications 5,6.
compound with antioxidant activitiy and has low
bioavailability and permeability values. The purpose One of the developments in the Drug Delivery
of this study is to knowing characterization nano- System in transdermal delivery is the vesicular
phytosomes of myricetin. system, it is known as phytosomes. Phytosomes are
Nano-phytosomes is made using thin-sonication a combination of phospholipids, such as
hydration method with a variation ratio of myrisetin: phosphatidylcholine in nonpolar solvents such as
phosphatidylcholine: cholesterol 1: 1: 0.4 (F1), 1: 2: acetone. In case, phytosomes are made up of
0.4 (F2) and 1: 3: 0.4 (F3). Characterization of nano- complex micellar structures of nature –
phytosome includes particle size, polydispers index,
phospholipids 7.
stability, adsorption efficiency and antioxidant
activity. The composition of phytosomes is safe and its
The results showed that particle size in F1, F2 and
components are accepted use for pharmaceutical,
F3 were 233.6 nm, 250.0 nm, and 242.7 nm with
polydispersion indexes were 0.260, 0.260 and 0.447, phytosome can increasing the absorption and
respectively. Potential zeta values (F1) -21.7 mV, (F2) bioavailability of water-soluble natural ingredients.
-15.7 mV and (F3) -11.3 mV. Entrapment efficiency This delivers a better therapeutic effect 8.
(F1) 91.94%, (F2) 91.39% and (F3) 91.39%. The
antioxidant activity of the formulas have values (F1) Nano-phytosomes are made by mixing
41,13 ppm; (F2) 25.46 ppm; and (F3) 20.64 ppm. phytoconstituents with phosphatidylcholine at
Based on the evaluation results, that the more the certain molar ratios (1: 1 to 1: 3), it will produce a
concentration of phosphatidylcholine added, the more complex with stronger bonds because
particle size, polydispersion index, adsorption phytoconstituent molecule will be bound by
efficiency and antioxidant activity were increases. phosphatidylcholine. The methods used in making
nano-fitosomes include solvent evaporation, reflux,
Keywords: nano-phytosome, myricetin, thin layer film
salting out, and lyophylization methods 9
hydration
The purpose of this study was to create a Drug
I. INTRODUCTION Delivery System nanoparticles with a vesicular
Flavonoid is a common group of polyphenol system of phytosomes from mirysetin powder using
and have many benefits such as antioxidants, three phosphatidylcholine comparisons.
antimicrobials, anticancer and anti-inflammatory II. MATERIAL AND METHOD
effects 1 . Mirycetin is a natural polyphenol
flavonoid that is widely distributed in fruits, UV-Vis Spectrophotometer (Genesys 10s,
vegetables and herbs that are being studied use for Thermo scientific), rotary evaporator (Heidolph),
treatment such as antioxidants. Mirycetin like other probe sonicator (QSonica, Newtown, USA),
flavonoids has limitations in bioavailability and particle size analyzer (Malvern Panalytical, USA),
absorption 2-4 . Low absorption due to its magnetic stirer (Thermo Scientific Scientific) ,
solubility in lipids is so bad and it need to make a China), centrifuges (SPLC Series, Gemmy 8 Hole,
new formulations for mirysetin. Taiwan), analytical scales (Ohaus), glassware
(Pyrex, Japan) and non-glass found in the
laboratory.

Copyright © 2020 The Authors. Published by Atlantis Press SARL.

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The sample were myricetin (Tocris, China), Vis spectrophotometry at 369 nm wavelength. The
Phospholipon 90 G (Lipoid, Germany), cholesterol entrapment efficiency (% EE) is calculated by the
pa (Sigma, USA), acetone pa (Merck), ethanol pa formula:
(Merck), dichloromethan pa (Merck), aqua pro
injection (PT. Ikapharmindo Putramas).
A. Nano-phytosome Formulation ........................... (1)

Nano-phytosomes are formulated by making TD is the total number of myricetin contained

three different variations using the sonication thin in the formula and FD is the number of myricetin
layer film hydration method. The nano-phytosome detected in the supernatant (not absorbed).
formula can be seen in table.
3. Nano-phytosome Stability
TABLE 1. NANO-PHYTOSOME FORMULA OF
MYRICETIN Nano-phytosome of myricetin was evaluated
after storage at 3 weeks of storage at 27o C. During
Myricetin : Phosphatidylcholine: storage, we observed of separation phase, physical
Cholesterol and chemical changes of the preparations were
Materials
F1 F2 F3 made.
(1:1:0.4) (1:2:0.4) (1:3:0.4)
Myricetin (mg) 10 10 10
Phosphatidylcholine 24 48 71
4. Antioxidant Activity
(mg)
Cholesterol (mg) 2 2 2 0.1 mL of 0.4 M DPPH solution was mixed
Acetone (ml) 20 20 20 with 1.0 mL of each concentration series of the test
Dichloromethane (ml) 5 5 5 solution. Then each mixture was vortexed for 30
Aqua pro Injection (ml) 25 25 25 seconds and left for OT (Operating Time (45
*In molar comparison minutes)). Then measured the solution for its
absorbance at a maximum wavelength (516 nm).
Nano-phytosome is made by dissolving Absorbance measurements were performed on pure
myricetin and phosphatidylcholine in acetone p.a, myricetin and nano-phytosome of myricetin
10 ml each ingredient, and cholesterol is dissolved samples.
in dichloromethane p.a. Phytoactive and
phospholipid solutions were mixed using a
magnetic stirrer at 35° C with rotation of 2000 rpm
III. RESULT AND DISCUSSION
in 10 minutes, the nano-phytosome complex was
A. Particle Size Analysis
made a thin layer film on a rotary evaporator at 55°
C and speed 50 rpm until the solvents evaporated. Particle size is the most important characteristic
The thin layer film formed on the walls of the in a nanoparticle system because it determines the
round flask then hydrated with aqua pro injection speed and ease of the drug to optimally absorbed.
characterized by the formation of colloidal The phospholipids will influences the particle size
dispersions. Colloidal dispersions formed were and the stability of the nanoparticle dispersion. The
sonicated using probe sonication for 10 minutes results nano-phytosome of myricetin particle size
with an amplitude of 60%. analysis showed that formula 1 to 3 had fulfilled
B. Characterization of nano-phytosome the nanoparticle size range of 10-1000 nm 10.
1. Determination of Zeta Potential and The results of the particle size analysis are shown
Particle Size Distribution in table 2.

To find out the size of nano-phytosome, particle TABLE 2. RESULTS OF MYRICETIN NANO-
size analysed and particle size distribution were PHYTOSOME CHARACTERIZATION
carried out using a Particle Size Analyzer (PSA). Formula
Evaluation
To find out the zeta potential value were using the 1 2 3
zeta potential analyzer. Particle size (nm) 233,6 250,0 242,7
1,209 3,552 1,792
2. Determination of Entrapment efficiency Polydispersity Index
0,260 0,260 0,447
0,001 0,008 0,006
Nano-phytosome myricetin was centrifuged at Potential Zeta (mV)
-21,7 -15,7 -11,3
3000 rpm at room temperature (27 ° C) for 50 1,209 0,750 0,624
minutes in order to separate the not absorbed active The results showed that formula 1 with a
substance. The supernatant from centrifugation comparison of the concentration of myricetin:
from formulas 1, 2 and 3 was taken 0.5 ml each, phosphatidylcholine: cholesterol (1: 1: 0.4) had the
then diluted with aqua pro injection up to 10 ml, smallest average particle size that is 233.6 nm. The
then the absorption was read three times using UV- concentration of phosphatidylcholine is bigger than
myricetin can bind myricetin perfectly because 1

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phytoconstituent molecule will be bound by 1 TABLE 3. STABILITY NANO-PHYTOSOMES

phosphatidylcholine molecule so the bond is OF MYRICETIN AT ROOM
stronger and the particle size can increase. Adding TEMPERATURE.
cholesterol to the formula can increase the physical
stability of nano-phytosome for more than 21 days. Formula 1st week 2nd week 3rd week

B. Polydispersity Index (IP) 1 No Sediment No Sediment No Sediment

IP is a value that explain the extent of particle 2 No Sediment No Sediment Sediment

size distribution in a preparation. IP for 3 No Sediment Sediment Sediment
monodispersion particles has a value of ≤0.5, while
IP >0.5 represents a nanoparticle system with a There is sediment in 2 and 3 formula, the
very wide particle size distribution sediment is reversible because it can be dispersed
(polydispersion). The best polydispersity index quickly again after shaking. Formula 1 does not
value is <0.5 because the smaller the IP value, the undergo precipitation and remains clear until 3rd
better the stability of nano-phytosome. The weeks.
polydipersity index results are shown in table 2.
E. Entrapment Efficiency
The smallest polydispersity index value is
shown in formulas 1 and 2 that is 0.260. The The aims of entrapment efficiency to determine
smaller polydispersity index value said to say the the amount of myricetin that absorbed in the nano-
preparation has a homogeneous distribution of phytosome carrier system. Determination of levels
particles with other particles, this shows that the of active substances that are not absorbed is
myricetin nano-phytosome is homogeneous and has calculated using the equation:
a monodispersion particle system.
Y = 1,057x10-3 + 0,0603 . x .............. (2)
C. Potential Zeta
The results of the adsorption efficiency are
Zeta potential is a measure of the magnitude of shown in table 4.
the electrostatic charge of particles in dispersion.
Zeta potential is measured to determine colloidal TABLE 4. RESULTS NANO-PHYTOSOM OF
stability. The colloidal solution system is stabilized MYRICETIN SAMPLE ANALYSIS
by the electrostatic repulsion force, where the Formula
greater the repulsive force between particles will Analysis Result
cause the particles to be difficult to close together 1 2 3
to form aggregates. Zeta potential value ± 30 mV Entrapment Efficiency (%) 91,94 %, 91,39% 91,39%
has good colloidal stability. The zeta potential
results are shown in table 2. Antioxidant activity (ppm) 41,13 25,46 20,64

The best measurement results of zeta on

myricetin nano-phytosome in formula 1 of three
The results of the adsorption efficiency in
replications has an average value of -21.7 mV,
negative results indicate that the formula 1 were 91.94%, it means that myricetin
was 91,94% absorbed in the phospholipid
phosphatidylcholine used is negatively charged.
component, formula 2 and 3 were 91.39%
D. Nano-phytosome Stability myricetin was absorbed in the phospholipid
component. From these results, each formula have
Nano-phytosome of myricetin is stored at 27°C a good entrapment efficiency range more than 80%.
for more than 3 weeks. The color nano-phytosome The difference in entrapment efficiency of each
of myricetin from fisrt week until third week has formula is due to differences in the amount of
the same color, it is yellowish. The smell that is phospholipids used. The more phosphatidylcholine
owned is the typical odor of myricetin. The results added into the formula, the efficiency of nano-
nano-phytosome of myricetin stability are shown in phytosome absorption was decrease. The highest
table 3. value of nano-phytosome of myricetin formula
entrapment efficiency is F1 which has 24 mg of
phosphatidylcholine has a particle size at 233.6 nm
and can absorb myricetin quite large at 91.94%.
F. Antioxidant Activity test
The antioxidant activity test was carried out
using the DPPH 1,1-diphenyl-2pikrilhidrazil
method (α, α-diphenyl-β-picrilhidrazil). DPPH is a

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free radical that is stable and does not form dimers 4. Wang L, Wang B, Li H, Lu H, Qiu F, Xiong L, et al.
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through UV-Vis spectrophotometry at a Kulkarni Walter E.R. Biodegradable polymeric
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antioxidant activity on myricetin because it has 2:224‐ 228.
22.69 ppm of IC50 value. IC50 value of myricetin F1 9. Freag MS, et al. Lyophilized Phytosomal Nanocarriers as
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IV. CONCLUSION

Characterization and evaluation nano-

phytosome of myricetin seen from the particle size
of all formulas having a size between 10-1000 nm,
the use of phosphatidylcholine at a concentration of
24 mg was able to produce the smallest particle
size is 233.6 nm and the lowest polydipersity index
is 0.260 with the highest entrapment efficiency is
91.94 %. Nano-phytosome of myricetin F2 and F4
are unstable for 3 weeks of storage. F1 has a zeta
potential value -21.7 mV, so F1 with the ratio of
myricetin: phosphstidylcholine : cholesterol (1 : 1 :
0.4) is the best formula in the production nano-
phytosome of myricetin.

ACKNOWLEDGMENT
The researchers say thanks to the Ministry of
for Research, Technology, and Higher Education of
the Republic of Indonesia who had funded this
research.

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