Nghiên cứu chế tạo, xác định hình thái, cấu trúc, tính chất các hệ nano chứa một số hợp chất lycopen, resveratrol và pycnogenol TT TIENG ANH

MINISTRY OF EDUCATION
AND TRAINING

VIETNAM ACADEMY OF
AND TECHNOLOGY

GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY


HO THI OANH

STUDY ON FABRICATION, DETERMINATION OF
MORPHOLOGY, STRUCTURE, AND PROPERTIES OF
NANOSYSTEMS CONTAINING SOME LYCOPENE,
RESVERATROL, AND PYCNOGENOL COMPOUNDS
Major: Organic Chemistry
Code: 9.44.01.14

SUMMARY OF CHEMICAL DOCTORAL THESIS

HA NOI – 2021


This thesis was completed at the Graduate University of Science
and Technology/Vietnam Academy of Science and Technology

Supervisors:
Dr. Hoang Mai Ha
Dr. Dang Thi Tuyet Anh

Examiner 1:

Examiner 2:
Examiner 3:

The thesis defense was monitored by the Graduate University level
Board of Examiner, held at Graduate University of Science and
Technology – 18 Hoang Quoc Viet – Cau Giay – Ha Noi.

Hardcopy of the thesis can be found at:
- Library of Graduate University of Science and Technology
- National Library of Vietnam


INTRODUCTION
1. The urgency of the thesis
Nowadays, following the trend of using natural resources of raw
materials, active ingredients with high biological activity such as lycopene,
resveratrol, pycnogenol,... are interested in scientists around the world in
research, extraction and widely application.
Although there are many valuable biological activities, most of the
above-mentioned active ingredients are classified as active ingredients with
very low water solubility and are difficult to absorb effectively into the
body, so their bioavailability is limited. To overcome the disadvantages of
solubility and durability, the research and manufacture of natural
biologically active substances in the form of nanoparticles promise to create
valuable sources of medicinal herbs for the pharmaceutical and cosmetic
industries.
On the other hand, the combination of active ingredients for the
combined product has a synergistic effect, providing higher biological
activity than using each active ingredient separately. Resveratrol and
pycnogenol, are known to be biologically active substances that have the

effect of increasing the stability and the solubility in water for carotenoids
with many conjugated double bonds such as lycopene. However, so far,
researchers have only stopped at making composites of active ingredients
without any published work on the fabrication of nanosystems, especially
lycopene/resveratrol and lycopene/pycnogenol nanosystems. Researching
and manufacturing these two nanosystems is a new research direction.
Therefore, the implementation of the thesis: "Study on fabrication,
determination of morphology, structure, and properties of nanosystems
containing some lycopene, resveratrol, and pycnogenol compounds" is
topical with a scientific significant study and practice.
2. Research objectives of the thesis
- Extraction of lycopene from Gac fruit, resveratrol from Reynoutria
1


Japonica Houtt., and synthesis of PLA-PEG copolymer as raw materials for
the fabrication of nanoparticles.
- Fabrication of nanoparticles and nanoparticle systems such as
nanolycopene, nanoresveratrol, lycopene/resveratrol nanosystem, and
lycopene/pycnogenol nanosystem as well as determination of the
morphology and structure of the obtained nanomaterials.
- Evaluation of the durability of nanomaterials under different
environmental and storage conditions.
3. The main research contents of the thesis
- Study on extracting lycopene from Gac fruit and resveratrol from
Reynoutria Japonica Houtt. Research on suitable conditions and methods of
extracting active ingredients. Determination of structure and purity of
lycopene and resveratrol after extraction.
- Research on synthesizing PLA-PEG copolymer used as encapsulation
agent for composite nanosystems containing lycopene.

- Research and fabrication of nanolycopene, nanoresveratrol with good
dispersion in water. Determination of morphology, structure, and property
of nano samples by modern analytical methods. Investigation of the
durability of nanopowder in different storage and environmental conditions.
- Research and preparation of lycopene/resveratrol nanosystem and
lycopene/pycnogenol nanosystem. Determination of morphology, structure,
and properties of nano samples by modern analytical methods. Studying the
stability of lycopene in nanosystems over storage time under different
conditions.
CHAPTER 1. OVERVIEW
The overview consists of 3 main parts: Part 1 provides an overview of
the research subjects, which are natural bioactive substances such as
lycopene from Gac fruit, resveratrol from Reynoutria Japonica Houtt., and
pycnogenol from red pine. The process of collecting, studying, and
2


researching these active ingredients shows that their bioavailability is
limited due to their poor solubility and durability. Nanotechnology is
considered an effective solution in improving dispersion, absorption,
enhancing medicinal properties, and durability of natural bio-active
ingredients. Therefore, section 2 provides an overview of research methods
for the fabrication of nanoforms of natural active ingredients. Part 3
presents the research situation in the country and the world on the
nanoparticle form of bio-active substances, namely nanolycopene,
nanoresveratrol,

lycopene/resveratrol

nanosystem,

and

lycopene/pycnogenol nanosystem.
CHAPTER 2. EXPERIMENT AND METHODOLOGY
2.1. Research Subjects
2.1.1. Materials and Chemicals
2.1.2. Equipment
2.2. Experiment
2.2.1. Extraction of natural compounds lycopene and resveratrol, used as
raw materials for the fabrication of their nanoparticle forms
2.2.2. Synthesis of PLA-PEG copolymer
2.2.3. Research and fabrication of nanosystems containing compounds of
lycopene, resveratrol, and pycnogenol
2.2.3.1. Preparation of nanolycopen và nanoresveratrol
Lycopene or resveratrol with different concentrations and surfactants
Tween 80 and cremophor RH40 were completely dissolved in an organic
solvent by magnetic stirrer for 30 mins. The solution was then slowly added
dropwise to 100 ml of distilled water containing PEG 6000 and stirred at
9000 rpm for 15 mins to obtain a homogeneous mixed solution. Continue to
create nanopowder by spray drying method. Install the machine and set the
running program for the machine, collect samples, and clean the machine
after spray drying. The nano-products obtained were in the form of fine and
3


loose powder. The resulting lycopene nano has a bright red color, and the
resulting resveratrol nano has an ivory white color.
2.2.3.2. Preparation of nanosystems containing lycopene
 Preparation of lycopene/resveratrol nanosystem

Lycopene and resveratrol were mixed with different ratios of 1:2, 1:1,
and 2:1, respectively, then added surfactants and synthetic copolymer
microencapsulated above, dissolved components in the tetrahydrofuran
solvent using an Ultra-Turrax homogenizer at 8400 rpm for 45 minutes;
The mixture after completely dissolving was slowly added to the distilled
water solution while stirring at a speed of 9000-10000 rpm for 30 minutes.
The lycopene/resveratrol nanosystem solution in water was then put into the
spray dryer system, resulting in lycopene/resveratrol nanopowder with a
plum red color.
 Preparation of lycopen/pycnogenol nanosystem
Lycopene and pycnogenol were mixed with different ratios, then added
surfactant Tween 80, components were completely dissolved in
tetrahydrofuran (THF) solvent by a magnetic stirrer, for 60 min. The
obtained solution was slowly added to the distilled water solution
containing PLA-PEG copolymer under stirring conditions at 9000 rpm for
30 minutes. Next, the homogenous mixture was put into a rotary vacuum
distillation system to remove THF solvent. The lycopene/pycnogenol
nanosystem in water was then cooled at -54oC. After 24 hours of freezing,
the lycopene/pycnogenol sample was transferred to the lyophilization
equipment system. Finally, the lycopene/pycnogenol nanopowder is fine,
loose, and bright red.
2.2.4. Research on nanopowder generation methods
- Spray-drying method.
- Freeze-drying method.
2.2.5. Quantitative analytical methods
Quantification of lycopene and resveratrol by HPLC high-performance
4


liquid chromatography and UV-Vis absorption spectroscopy.

2.2.6. Evaluation of the durability of nanoparticles
- Investigating the change of active ingredient content of nanoparticles.
- Studying on morphological changes of nanoparticles.
2.2.7. Methods for analyzing the structure and properties of materials
- The structures of the materials were determined by infrared (FT-IR)
and nuclear magnetic resonance (NMR) spectroscopy.
- Molecular weight of PLA-PEG copolymer was determined by GPC.
- The particle size distribution of nanopowder products was studied by
the dynamic light scattering method (DLS).
- Morphology and size of nanomaterials samples were evaluated by
transmission electron microscopy TEM.
CHAPTER 3. RESULTS AND DISCUSSION
3.1. Results of extraction of lycopene, resveratrol, and synthesis of
PLA-PEG copolymers, used as raw materials for the fabrication of
nanosystems.
3.1.1. Extraction of lycopene compounds from gac aril
3.1.1.1. Determine the appropriate temperature and time for the drying
process of gac aril
Research results show that the appropriate temperature and time for
drying gac aril is 60oC within 15 hours.
3.1.1.2. Determination of suitable organic solvents to extract lycopene from
dried gac aril by Soxhlet method
The active substance dissolves best in chloroform solvent, then
gradually decreases from tetrahydrofuran, dichloromethane to hexane.
However, due to the lowest toxicity, dichloromethane was chosen as the
suitable solvent for lycopene extraction.
3.1.1.3. Evaluation of the structure and purity of the extracted lycopene
a) The structure of the extracted lycopene was analyzed by FT-IR
5

spectroscopy
On the FT-IR spectrum, there are absorption bands characteristic of
functional groups present in the molecule. The waveband appearing at 3038
cm-1 is typical for the valence vibration of the =CH (alkene) bond, at 2912
cm-1 and 2854 cm-1 is for the valence vibration of the =CH (alkane) bond. .
The characteristic valence oscillation of the C=C (alkene) bond occurs at
1626 cm-1. The bands appearing at 1439 cm-1 and 1390 cm-1 are the
characteristic absorption bands for the deformation vibrations of the -CH2
and -CH3 groups, respectively. In particular, the strongly absorbed
waveband at 959 cm-1 characterizes the out-of-plane strain vibration of =CH
(trans-alkene).
b) The structure of the extracted lycopene was analyzed by NMR
spectroscopy

Hình 3.6. The structural formula of lycopene
1

On the H-NMR spectrum, there are resonance signals characteristic of
protons present in the molecule. The resonance signal appearing at the
weakest field δ=6.66-6.60 ppm (m, 2H) is attributed to the proton H-7&H11. Other alkene protons are specifically attributed as follows: δ=6.49 ppm
(dd, J=11.0; 15.0 Hz; 1H; H-12); 6.35 ppm (d; J=15.0 Hz, 1H; H-8); 6.266.19 ppm (m; 2H; H-15&H-14); 6.18 ppm (d; J=11.5 Hz; 1H; H-10); 5.95
ppm (d; J=11.0 Hz; 1H; H-6); 5.12-5.09 ppm (m; 1H; H-2). The other
protons of the alkane fraction are specifically attributed as follows: 2.12
ppm (d; J=4.5 Hz; 4H; H-4&H-3); 1.97 ppm (s; 6H; 2CH3); 1.82 ppm (s;
3H; CH3), 1.69 ppm (s; 3H; CH3), 1.61 ppm (s; 3H; CH3).
The

13

C-NMR spectrum shows the characteristic resonance signals of
6


the equivalent carbon atoms present in the molecule. The resonant signal
appearing at the weak field δ=139.5 ppm is assigned to C-5. The resonance
signals of the alkene carbon atoms are attributed as follows: δppm: 137.4
(C-13); 136.5 (C-9); 136.2 (C-1); 135.4 (C-12); 132.7 (C-15); 131.6 (C-8);
130.1 (C-14); 125.8 (C-7); 125.2 (C-6); 124.0 (C-2); 40.2 (C-4); 26.7 (C-3);
25.6 (CH3); 17.7 (CH3); 17.0 (CH3); 12.9 (CH3); 12.8 (CH3).
Comparing the obtained data with the standard spectrum of lycopene
shows that the signals appear to be completely consistent, showing that the
product after extraction is lycopene.
c) Lycopene quantification by absorption spectroscopy (UV-Vis)
UV-Vis spectra show that at the same concentration, the absorbance
band at 517 nm of the extracted lycopene sample is about 1% higher than
that of Sigma-Aldrich's 98% pure lycopene standard sample. Thus, it can be
concluded that the purity of the extracted lycopene sample is over 98%.
d) Lycopene quantification by high-performance liquid chromatography
(HPLC)
750

500

250

0
10

20

30

40

50

60

Figure 3.10. The extracted lycopene sample chromatogram was developed
with the solvent system MeOH: ACN: DCM (10:50:40)
Quantitative results by HPLC (Figure 3.10) showed that the extracted
lycopene reached a purity of > 98%, which is consistent with the
quantitative results by UV-Vis spectroscopy.
3.1.1.4. Lycopene storage
Lycopene degrades rapidly when stored at room temperature. At a
temperature of 4oC, corresponding to the temperature of the refrigerator
7


compartment, lycopene decomposes more slowly. At -16oC, lycopene is
relatively stable, the remaining lycopene content is 92% after 3 months of
storage. Therefore, the lycopene storage condition is to pack lycopene in
foil, vacuum it, and then store it at -16oC.
3.1.2. Study on extraction of resveratrol compounds from radix Polygoni
cuspidati
3.1.2.1. Structure analysis of resveratrol after extraction
a) Structure analysis of extracted resveratrol by FT-IR spectroscopy
The FT-IR spectrum shows resveratrol-specific signals. The sharpest
absorption band occurs at 3230 cm-1, which characterizes the valence

vibrations of the O−H (phenol) bond. The absorption band at 3021 cm-1
represents the valence vibration of the vinyl group (=C−H), at 2890 cm-1
represents the valence vibration of the =C−H (alkane) bond. The valence
oscillations ν(C=C) of the benzene ring are shown in the absorption bands
1609 cm-1, 1579 cm-1, 1510 cm-1, and 1447 cm-1. In-plane strain oscillations
of the OH group appear in the absorption band 1382 cm-1. The valence
vibration of the C-O bond (from the phenol group) is evident in the
spectrum at 1150 cm-1. In addition, the absorption waveband at 968 cm-1
characterizes the strain oscillation of the alternative C-H bond on C=C from
the trans orientation. The band at 833 cm-1 is the characteristic strain
vibration of the C-H bond of the benzene ring and at 673 cm-1 is the
waveband showing the out-of-plane strain γ(OH) vibration of the -OH
group.
b) Structure analysis of extracted resveratrol by NMR spectroscopy

Figure 3.15. The structural formula of resveratrol
The results of 1H-NMR proton nuclear magnetic resonance spectroscopy
8


(500MHz, DMSO-d6) analysis show that resveratrol's proton-specific
signals. The resonance signals in the δH region 6.11-7.38 ppm characterize
the phenolic structure of resveratrol. The proton signals appearing at δH 6.38
(d, J = 2.0; 2H) and 6.11 (t, J = 2.0; 1H) indicate the presence of a
structured aromatic ring. Symmetry is substituted at 3 positions 1, 3, and 5.
Besides, the appearance of a pair of signals belonging to the AA'-BB' spin
interaction system at δH 6.75 (d, J = 9.0); 2H) and 7.38 (d, J = 8.5; 2H)
represent the presence of a para-substituted benzene nucleus.
The

13

C-NMR spectrum shows the characteristic signals of C atoms.

Attribution of resonance signals and carbon atoms in resveratrol results in
signals of 14 carbon atoms, including 9 metin and 5 carbons. quaternary
carbon. The metin carbon values resonate at position δC 104.3 (C2, C-6);
101.7 (C-4); 127.8 (C-2′, C-6′); 115.5 (C-3′, C-5′); 127.8 (C-7) and 125.6
(C8). Five quaternary carbons occur at δC 139.2 (C-1); 158.4 (C-3, C-5);
28.1 (C-1′) and 157.1 (C-4′).
In addition, the interaction constant JH-7=16.5 Hz demonstrates that the
C7/C8 double bond has a trans configuration. Comparison of the obtained
data with the standard spectrum of resveratrol shows that the signals appear
to be completely consistent, indicating that the main extraction product is
trans-resveratrol.
3.1.2.2. Quantification of resveratrol by absorption spectroscopy (UV-Vis)
and high-performance liquid chromatography (HPLC)
a) Quantification of resveratrol by absorption spectroscopy (UV-Vis)
UV-Vis spectra showed that at the same concentration, the absorbance
band at 310 nm of the extracted resveratrol sample was about 2.7% lower
than that of Sigma-Aldrich's 95% purity standard resveratrol sample. Thus,
the purity of resveratrol after extraction and purification is over 92%.
b)

Determination

of

resveratrol

by

high-performance

liquid

chromatography (HPLC)
Quantitative results by HPLC (Figure 3.19) showed that the extracted
9


resveratrol reached a purity of 92.3%, which is consistent with the results of
resveratrol quantification by UV-Vis spectroscopy.
DAD1 B, Sig=310,4 Ref=360,100 (RESVERATROL\21061107.D)

9.331

mAU
mAU

310nm

700
700

600
500

500
500

400
400

300
300

200
200

100
100

00
0
0

5

5

10

10

15

15

20

20
min

25

Figure 3.19. The extracted resveratrol sample chromatogram was developed
with the solvent system MeOH – H2O + 0.1%Fa = 40 – 60 (v/v)
3.1.3. Synthesis results of PLA-PEG copolymers
Table 3.9. Molecular weights of PLA-PEG copolymer samples
Mẫu
Mw (Da)
PDI
PLA-PEG.1
8400
1,2
PLA-PEG.2
11600
1,4
PLA-PEG.3
17200
1,7
Table 3.9 shows that the obtained copolymer molecular weight increases
with increasing lactide/mPEG ratio. Polylactide is a hydrophobic polymer
so PLA-PEG.1 sample with molar ratio nPEG:nlactide = 1:20 has a molecular
weight of 8400 giving good water solubility suitable for fabrication of
nanoparticles. PLA-PEG.1 sample was then used to study and fabricate
composite nanosystems containing lycopene.
The infrared spectra show the characteristic absorption bands of PLA,
and PEG is both shown on the spectrum of the PLA-PEG copolymer. The
absorption band at 3444 cm-1 characterizes the valence vibration of the -OH

group in the PEG molecule. The wide waveband at 2877 cm-1 characterizes
the valence vibrations of C-H2, 1759 cm-1 respectively, characterizes the
valence vibrations of the C=O group in the PLA molecule. Two wavebands
1454 cm-1 and 1382 cm-1 are typical for group deformation vibrations,
waveband 1249 cm-1 is typical for group vibrations of C-O-H group, and

10

30


waveband 1097 cm-1 is typical for – OH group deformation vibrations.
The structure of the PLA-PEG copolymer was confirmed by 1H-NMR
13

and

C-NMR spectra measured in CDCl3. Proton nuclear magnetic

resonance spectroscopy shows proton-specific signals of the PLA-PEG
copolymer. The resonance signal and proton position in the PLA-PEG
copolymer are as follows:
-

The signal at δ=1.59 ppm is that of the CH3 group proton in PLA.

-

The signal at δ=5.03 ppm is that of the CH group proton in PLA.

-

The signal at δ=3.63 ppm is that of the CH2 group proton in PEG

The

13

C-NMR spectrum shows the characteristic signals of C atoms.

Attribution of the resonance signals to the carbon atoms in the PLA-PEG
copolymer gives the following results:
-

The signal at δ=169.5 is that of the C=O group carbon in PLA.

-

The signal at δ=77.2 is that of the O-CH2 group carbon in PEG.

-

The signal at =70.5 is that of the O-CH group carbon in PLA.

-

The signal at =16.7 is that of the CH3 methyl carbon in PLA.

3.2. The results of preparation of nanosystems containing some
lycopene, resveratrol, and pycnogenol compounds

3.2.1. Results of lycopene nanofabrication
3.2.1.1. Effect of surfactants on the fabrication of lycopene nanoparticles
12

Sự phân bố (%)

10
8
NLy21

6

NLy12

4

NLy1

2
0
0

50

100

150

200

250

Đường kính hạt (nm)

Figure 3.23. Particle size distribution pattern of lycopene nanoparticle
according to different ratios between lycopene and surfactant Tween 80
11


The samples NLy1, NLy12, and NLy21

with lycopene/Tween 80

content ratios of 1/1, ½, and 2/1, respectively, resulted in average particle
diameters of 55 nm, 65 nm, and 76 nm, respectively (Figure 3.23). The
particle size distribution pattern shows that sample NLy1 has a narrow
distribution and the average particle size is less than 100 nm. Therefore, a
lycopene/Tween 80 ratio of 1/1 was chosen for the lycopene
nanoformulation
To evaluate the effect of different surfactants on the lycopene
nanoparticle fabrication process, the surfactant Tween 80 in the
nanofabrication formula studied above was replaced with cremophor RH40
with a ratio of lycopene/RH40 is 1/1- this is the ratio of active
ingredient/surfactant content that has achieved the best nanoparticle size.
12

Sự phân bố (%)

10
8

NLy1

6

NLy11

4
2
0
0

50

100

150

200

250

300

350

Đường kính hạt (nm)

Figure 3.24. Particle size distribution pattern of lycopene nanoparticles
using cremophor RH40 and Tween 80 surfactants
The results of DLS measurement of the lycopene nano samples showed

that the particles in the sample NLy1 (using the surfactant Tween 80) and
the particles in the sample NLy11 (using the surfactant cremophor RH40)
had sugar. The average particle diameter is 55 nm and 51 nm, respectively
(Figure 3.24). There is no significant difference between these 2 samples.
However, Polysorbate 80 is a more common and cheaper surfactant than
cremophor RH40. Therefore, Tween 80 was selected as a surfactant for the

12


fabrication of nanoparticle systems.
3.2.1.2. Evaluation of water dispersibility of lycopene nanopowder samples
Table 3.10. Evaluation results of the water dispersibility of lycopene and

Samples
Lycopene
NLy1
(4% lycopene)
NLy3
(8% lycopene)
NLy5
(12% lycopene)

nanolycopene samples
Observe the bottom of the
Dispersibility in water
glass jar after 1 day
Very poor
Precipitate
Very good

Very clear

Very good

Very clear

good

Precipitation occurs

3.2.1.3. Structure analysis of nanolycopene samples
The FT-IR spectrum shows the characteristic wavebands of PEG: at
3438 cm-1 (-OH), 2886 cm-1 (CHstr(sp2)), or 1111 cm-1 characteristic of the
bonding between the C–C–O and C–C–H groups, are clearly shown in the
infrared spectra of NLy1, NLy3 and NLy5 samples. For the lycopene
characteristic wavebands: the absorption band at 2907 cm-1, 2850 cm-1
(CHstr(sp3)), 1670 cm-1, 1642 cm-1 (C=Cstr(trans)), 1443 cm-1 (CH2(strain
vibration)) and 963 cm-1 (CH(trans OOP)) were present in the lycopene
nanocomposites but not clearly. This is explained because the lycopene
content contained in the nanopowder sample is much less than the PEG
carrier content in the lycopene nanoparticle.
3.2.1.4.

Morphology

and

particle

size

distribution

of

lycopene

nanoparticles
Particle size distribution (Figure 3.28 a) shows that Nly1, NLy3, and
NLy5 samples (with lycopene content from 4-12%) have average particle
diameters of 55 nm, 135 nm, and 289 nm, respectively, and corresponding
to TEM image results (Figure 3.28 b, c, and d). The change in particle size
of the samples is directly proportional to the lycopene content present in the

13


nanopowder samples, which means that the increase in lycopene content
leads to a larger particle size. The TEM image shows that the lycopene
nanoparticles have a spherical shape, uniform distribution, and no clumping
phenomenon.

Figure 3.28. Size distribution of lycopene nanoparticles obtained by (a)
DLS and TEM images of (b)-NLy1, (c)- NLy3, and (d)- NLy5
3.2.1.5. Evaluation of the durability of nano lycopene
Table 3.11. Lycopene stability in nanopowder samples over storage time at

Samples
NLy1

NLy3
NLy5

0 day
100
100
100

room temperature
Stability (Remaining lycopene content (%))
15th day
30th day
60th day 90th day
90.2
80.4
70.2
60.1
91.3
82.7
73.6
64.5
92.4
84.9
76.9
69.0

At room temperature, the lycopene present in the nanopowder samples
was significantly degraded (Table 3.11). After 90 days of storage, the
remaining lycopene content in samples NLy1, NLy3, and NLy5 were
60.1%, 64.5%, and 69%, respectively. Thus, the fastest lycopene

degradation occurred in the NLy1 sample, corresponding to the lycopene
content in the sample of 4%, then the NLy3 sample (the lycopene content is
14


8%), and finally the lycopene degradation occurred slowly. the most in
sample NLy5 (the lycopene content is 12%).
3.2.2. Result of resveratrol nanofabrication
3.2.2.1. Evaluation of the water dispersibility of resveratrol nanopowder
samples
Table 3.12. Evaluation results of the water dispersibility of resveratrol and

Samples
Resveratrol
NR1
(10% resveratrol)
NR2
(15% resveratrol)
NR3
(20% resveratrol)

nanoresveratrol samples
Dispersibility in
Observe the bottom of
water
the glass jar after 1 day
Very poor

Precipitate

Very good

Very clear

Very good

Very clear

Very good

Clear and without
precipitation

3.2.2.2. Structural analysis of nanoresveratrol
Some of the PEG-specific wavebands appear clearly in the FT-IR
spectrum: at 3438 cm-1 (-OH), at 2886 cm-1 (CHstr(sp2)), or 1111 cm-1
characteristic of the link between groups C–C–O and C–C–H, are clearly
shown in the infrared spectrum of the NR1, NR2, and NR3 samples. Some
characteristic wavebands of resveratrol are: absorption wavebands at 3279
cm-1 (-OH), 964 cm-1 (trans-olefinic), 1584 cm-1 (C=C), and 1145 cm-1 (CO) was present in resveratrol nano samples but was not clear. This is
explained because the resveratrol content contained in the nanopowder
sample is much less than the PEG carrier content in the resveratrol
nanoparticle.
3.2.2.3. Morphology of resveratrol nanoparticles
The particle size distribution in Figure 3.32a shows that the NR1, NR2,
and NR3 nanoparticles have average particle diameters of 12 nm, 19 nm,
and 38 nm, respectively, corresponding to the TEM images (Figure 3.32b,
15

c, and d). Increasing resveratrol content leads to an increase in the size of
nanoparticles. The TEM image (Figure 3.32b) shows that the nanoproduct
containing 10% resveratrol (NR1) has a spherical shape, with small particle
sizes in the range of 10-18 nm. Similar to Figures 3.32c and d, the
nanoparticle size ranges from 14-21 nm (NR2 sample), 35-42 nm with NR3
sample. The particle morphology and particle size distribution of the
nanopowder samples completely corresponded with the sensory evaluation
of the water dispersion of the obtained powder samples.

Figure 3.32. Size distribution of resveratrol nanoparticles obtained by (a)
DLS and TEM images of (b)-NR1, (c)- NR2, and (d)- NR3
3.2.2.4. Investigation of the stability and morphology of resveratrol
nanoparticles in different pH environments
Research results show that nano resveratrol is relatively stable in an
acidic environment, the decomposition rate of resveratrol nano is increased
rapidly in alkaline environments. Specifically, at pH 4.5 and 7, the
remaining resveratrol content was 90% and 62.8%, respectively after 1 day.
However, at pH 8 the resveratrol content remaining after 1 day was 51.4%
and at pH 9 the remaining resveratrol content after 180 minutes was 20.5%.
Meanwhile, according to research by Š. Zupančič, Z. Lavrič, and J. Kristl,

16


at pH 9, trans-resveratrol was completely degraded within 10.1 min. Thus,
the preparation of resveratrol in the form of nanoparticles has improved the
stability of resveratrol. This is explained by the role of surfactant tween 80
and encapsulation PEG 6000 to help protect resveratrol from the effects of
alkaline environments.
3.2.3. Results of preparation of lycopene/resveratrol nanosystems

3.2.3.1. Structure analysis of lycopene/resveratrol nanosystems
The characteristic absorption band of the -OH group in the FT-IR
spectrum of resveratrol is at 3250 cm-1. However, in the FT-IR spectra of
the system S1, S4, and S7, it shows that the -OH group of resveratrol has
been elongated and moved to the vibrational positions of 3365 cm-1, 3398
cm-1, 3375 cm-1, respectively, which indicates that an intermolecular
hydrogen bonding interaction occurs between resveratrol and the PLA-PEG
surfactant/copolymer. Besides, the absorption wavebands of lycopene in the
S1, S4, and S7 nanosystems in the FT-IR spectrum are also oscillating
compared with the characteristic bands in the FT-IR spectrum of lycopene.
For example, the CHstr(sp2) oscillations of lycopene in the nanosystem tend
to shift close to the wavelengths 2923 cm-1, 2924 cm-1, 2922 cm-1 while this
oscillation is in the FT-IR spectrum of pure lycopene. purity is 3038 cm-1.
The position change of these absorption bands indicates good compatibility
between lycopene and PLA-PEG copolymer. Another example also shows
the C=Cstr(trans) oscillations of lycopene in the S1, S4, and S7 nanosystems
at wavelengths 1593 cm-1, 1604 cm-1, 1604 cm-1 respectively during this
oscillation. of pure lycopene is 1625 cm-1. This phenomenon is due to the
extra molecular interaction between lycopene and resveratrol.
The structure of the nanosystem was also analyzed by UV-Vis
spectroscopy. The characteristic absorption bands of resveratrol in
nanosystems appear at 295 nm and 310 nm, respectively. The three
characteristic absorption peak wavebands of lycopene in the nanosystem are
450 nm, 479 nm, and 510 nm, respectively. The UV-Vis spectrum shows
17


that the absorption intensity of the resveratrol and lycopene peaks in the
samples is proportional to the ratio of the content of two active ingredients
resveratrol and lycopene present in the lycopene/resveratrol nanosystem.

3.2.3.2. Morphological characteristics and particle size distribution of
lycopene/resveratrol nanosystems
The particle size distribution pattern of samples S1, S4 and S7 in water
shown in Figure 3.39a shows that the average particle diameters of the
samples are 66 nm, 79 nm, and 102 nm, respectively. TEM images (Figures
3.39b, 3.39c, and 3.39d) show that the obtained nanoparticles have a
spherical shape. The particle size is proportional to the lycopene/resveratrol
ratio. The good compatibility between lycopene and resveratrol during the
fabrication process gave the nanoparticle system a relatively uniform
distribution, the particle size being less than 100 nm, although the total
content of two active ingredients lycopene and resveratrol was up to 12%.

Figure 3.39. Size distribution of lycopene/resveratrol nanoparticles
obtained by (a) DLS and TEM images of (b)- S1, (c)- S4, and (d)- S7
The results of the morphological evaluation and particle size distribution
also showed that the combination of natural active ingredients for
composite

products

had

synergistic

18

and

optimal

effects.


Lycopene/resveratrol nanosystem contains lycopene content of 4% and
resveratrol is 8%, ie the total content of the two active ingredients is up to
12%, the average particle size of the system still reaches 66 nm.
Meanwhile, for the lycopene nano samples made in Section 3.2.1, when the
lycopene content is over 10%, the nanomaterial size increases significantly
above 200 nm. For the resveratrol nano samples (Section 3.2.2), the average
particle size of the products was below 100 nm despite the resveratrol
content up to 20%. Thus, the lycopene/resveratrol nanoparticle system
showed very good compatibility between lycopene and resveratrol.
Resveratrol has shown the role of reducing the particle size and increasing
the uniform distribution of nanoparticles in the system.
3.2.3.3.

Evaluation

of

lycopene

stability

in

lycopene/resveratrol

nanosystems
The results show that the degradation rate of lycopene when storing

samples at -16oC (Table 3.14) is nearly half slower than the decomposition
rate of active ingredients at room temperature (Table 3.13). The remaining
lycopene content in the nanopowder samples S1, S4, and S7 was 94.8%,
93.1%, and 91.5%, respectively while the remaining lycopene content in
these samples at room temperature was 88.8%, 85.0%, 83.2%, respectively.
Thus,

the

appropriate

storage

conditions

for

lycopene/resveratrol

nanosystems are in a cold environment of -16oC.
Table 3.13. Lycopene stability in lycopene/resveratrol nanosystem powder
samples over storage time at room temperature
Stability (Remaining lycopene content (%))
Samples
0 day
15th day
30th day
60th day
90th day
S1

100
97.3
94.6
91.8
88.8
S2
100
97.9
95.7
93.2
90.8
S3
100
97.5
95.0
93.0
90.5
S4
100
96.5
92.9
89.0
85.0
S5
100
97.2
94.2
90.2
86.8
S6

100
97.0
94.3
90.1
86.2
19


S7
S8
S9

100
100
100

96.0
96.6
96.8

91.9
92.2
92.5

87.6
88.8
90.0

83.2
84.6

84.8

Table 3.14. The stability of lycopene in lycopene/resveratrol nanopowder

Samples
S1
S2
S3
S4
S5
S6
S7
S8
S9

samples over storage time at -16oC
Stability (Remaining lycopene content (%))
0 day
100
100
100
100
100
100
100
100
100

15th day
99.2

99.5
99.4
98.8
99.1
99.2
98
98.3
98.4

30th day

60th day

98.2
98.8
98.4
97.4
98.0
98.1
96
96.5
96.7

96.5
97.2
96.9
95.3
96.1
96.2
93.8

94.7
94.9

90th day
94.8
95.6
95.3
93.1
94.2
94.1
91.5
92.8
93.0

3.2.4. Results of preparation of lycopene/pycnogenol nanosystems
3.2.4.1. Structure analysis of lycopene/pycnogenol nanosystems
The FT-IR spectrum clearly shows the characteristic absorption
wavebands for the vibrations of lycopene, pycnogenol and copolymers in
samples S1, S2, and S3. Specifically: the absorption waveband in the region
3600-2500 cm-1 is typical for the -OH valence vibration of pycnogenol and
PEG molecule (in PLA-PEG copolymer), the absorption waveband at 1100
cm-1 (CH(trans)) of lycopene, for the CHstr(sp2) valence vibration of
lycopene at 2912 cm-1 there is a slight shift in the FT-IR spectra of the
product samples (for S1 sample waveband at 2913 cm-1, S2 sample at
2916 cm-1 and S3 sample at 2915 cm-1). The characteristic absorption band
for the C=C (aromatic ring) valence vibration at 1610 cm-1 of pycnogenol is
evident in the spectra of S1, S2, and S3 samples. In addition, it can be
noticed that the characteristic wavebands of PLA-PEG copolymer
microencapsulation agent such as 2875 cm-1 (CH2 valence vibrations in
PLA molecule), 1759 cm-1 (chemical fluctuations) values of the C=O group

20


in PLA) and 1454 cm-1 (CH strain fluctuations) are most clearly shown on
the infrared spectrum of the samples.
3.2.4.2. Evaluation of the dispersion ability of lycopene/pycnogenol
nanopowder samples
The good compatibility between lycopene and pycnogenol with tween
80 surfactant and PLA-PEG copolymer carrier resulted in a nanopowder
product with very good dispersion in water and no sign of aggregation after
many days of storage. managed under normal conditions. Specifically, the
complete self-dispersion time in water of 150 mg of each powder sample
S1, S2, and S3 was 1 min, 2 min, and 3.5 min, respectively.
Lycopene/pycnogenol nano samples with a lycopene/pycnogenol content
ratio of 1/2 gave the clearest red solution and the clarity gradually
decreased as the lycopene/pycnogenol content ratio increased.
3.2.4.3. Morphology of lycopene/pycnogenol nanopowder samples

Figure 3.45. Size distribution of lycopene/pycnogenol nanoparticles
obtained by (a) DLS and TEM images of S1, S2, and S3.
The particle size distribution plot shows that samples S1, S2, and S3
have average particle diameters of 73 nm, 85 nm, and 114 nm, respectively,
corresponding to the TEM image results (Figure 3.45). The change in
particle size of the samples is directly proportional to the ratio of
21


lycopene/pycnogenol content, which means that an increase of the
lycopene/pycnogenol ratio leads to increasing particle size. The TEM image
shows that the lycopene/pycnogenol nanoparticles are spherical, uniformly

distributed, and have no clustering phenomenon
3.2.4.4. Morphological evaluation of lycopene/pycnogenol nanoparticles in
different pH environments
The results of the study of particle morphology in the pH 5 and 9
environments showed that the pH environment influenced on the properties
of the nanoparticle system. Nano lycopene/pycnogenol are easily
agglomerated when dispersed in an acidic medium. In the basic
environment, the particle size and nanoparticle morphology did not change
much.
CONCLUSION
1.

The valuable active ingredients lycopene and resveratrol have been

extracted from Vietnamese herbal resources and successfully synthesized
PLA-PEG copolymers used as raw materials for the fabrication of
nanosystems containing some lycopene, resveratrol, and pycnogenol:
- Lycopene was extracted from the dried Gac membranes by the Soxhlet
method using dichloromethane as the organic solvent and purified by
ethanol. The amount of extracted lycopene is about 3.2-4.4 g/kg dry Gac
membrane. The obtained lycopene has a high purity of ≥ 98%.
- The active ingredient resveratrol has also been successfully isolated
from the rhizome roots by the penicillium strain fermentation method. The
amount of extracted resveratrol is about 3.0-4.5 g/kg of the rhizome root.
The obtained resveratrol has a high purity of over 92%.
- The PLA-PEG copolymer was successfully synthesized with an Mw
weight of 8400 and a PDI index of 1.2. This copolymer is used as a
microencapsulation agent for the fabrication of lycopene-containing
nanosystems.
22

2.

Four types of nano-products of bio-active ingredients have been

prepared, including nano lycopene, nano resveratrol, lycopene/resveratrol
nanosystem, and lycopene/pycnogenol nanosystem. All nanoforms were
obtained in the form of fine powders, which have very good dispersion in
water, the obtained nanoparticles are spherical and have a fairly equal
particle size distribution:
- Nano lycopene and nano resveratrol have been successfully prepared
by spray drying method using a surfactant Tween 80 (with an appropriate
active ingredient/surfactant ratio of 1/1) and microencapsulation agent is
PEG 6000.
+ Lycopene nano products have lycopene content from 4-12%
corresponding to the average particle size from 55-289 nm. The durability
of the lycopene nanopowder samples when stored at room temperature is
not high, the remaining lycopene content after 90 days for the lycopene
nano sample (containing 4% lycopene) is 60.1%, and for the lycopene nano
sample (containing 8% lycopene) is 64.5% and for samples containing 12%
lycopene, it is 69.0%.
+ The average particle size of the resveratrol nanoparticle samples is
from 12-38 nm, the particle size is proportional to the ratio of lycopene
content present in the nanopowder sample. The stability and particle
morphology of the resveratrol nanoparticle changed significantly when
dispersed in different pH media. At pH 4.5, the nanoparticles were easily
agglomerated, the particle diameter increased from 12 nm up to 85 nm. At
pH 9, the nanoparticles decomposed rapidly, after 180 minutes the
remaining resveratrol content in the nano sample was only 20.5%.

- Lycopene/resveratrol nanosystems with ratios of 4% lycopene/8%
resveratrol, 6% lycopene/6% resveratrol, and 8% lycopene/4% resveratrol,
respectively, have been successfully prepared by the spray-drying method.
The obtained product has good dispersion in water, the particles are
spherical, the particle size is small in the range from 66-102 nm, and very
23