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 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 Research objectives of the thesis - Extraction of lycopene from Gac fruit, resveratrol from Reynoutria 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 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 OVERVIEW The overview consists of main parts: Part 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 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 provides an overview of research methods for the fabrication of nanoforms of natural active ingredients Part 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 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 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 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 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 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 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 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 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 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 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 compartment, lycopene decomposes more slowly At -16oC, lycopene is relatively stable, the remaining lycopene content is 92% after 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 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 NLy21 NLy12 NLy1 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 NLy1 NLy11 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 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 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 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 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 day However, at pH the resveratrol content remaining after day was 51.4% and at pH 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 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 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 (%)) 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 min, 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 and 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 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 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 equally distributed After 90 days of storage at -16oC, the lycopene/resveratrol nanosystem product (lycopene/resveratrol content is ½) in the presence of the antioxidant vitamin E showed high durability The remaining lycopene content after months reached 95.6% - Lycopene/pycnogenol nanosystems with ratios of 4% lycopene/8% pycnogenol, 6% lycopene/6% pycnogenol, and 8% lycopene/4% pycnogenol, respectively, were successfully prepared by the freeze-drying method The obtained nanoparticles have average particle sizes in the range of 73-114 nm The nanoparticle morphology in the pH environment resulted in the lycopene/pycnogenol nanoparticle being easily aggregated, leading to increased particle size Meanwhile, in the pH environment, the nanoparticles were relatively uniformly distributed, there was no clumping phenomenon and the average particle diameter did not change much compared to the original NEW CONTRIBUTIONS OF THE THESIS A process of extracting and purifying lycopene from Gac membranes has been developed by a simple, safe, and easy to deploy method on an industrial scale Lycopene obtained with high purity of over 98% can be used as raw materials for pharmaceutical and functional food industries Two lycopene nanosystems including lycopene/resveratrol nanosystem and lycopene/pycnogenol nanosystem are two new nanomaterial systems Nanopowder products have good dispersion in water, small particle size below 100 nm, and high strength These products have potential applications as raw materials for domestic pharmaceutical companies The extraction of lycopene with a high purity of over 98% and the successful manufacture of lycopene-containing nanopowder from the source of Gac fruit will contribute to improving the economic value of Vietnamese Gac fruit 24 LIST OF THE PUBLISHED WORKS Ho Thi Oanh, Nhung Hac Thi, Thanh Nhan Nguyen, Tuyet Anh Dang Thi, Tuyen Van Nguyen, and Mai Ha Hoang, Co-encapsulation of lycopene and resveratrol in polymeric nanoparticles: Morphology and lycopene stability, Journal of Nanoscience and Nanotechnology, 21(5), 3156-3164, 2021 Hoang Mai Ha, Dang Thi Tuyet Anh, Ho Thi Oanh, Hac Thi Nhung, Nguyen Duc Tuyen, Nguyen Van Tuyen, The extraction method of pure lycopene directly from Gac aril, Patent No.27630, 2021 Hoang Mai Ha, Dang Thi Tuyet Anh, Ho Thi Oanh, Hac Thi Nhung, Nguyen Duc Tuyen, Nguyen Van Tuyen, The method of producing lycopene/resveratrol nanosystem, Patent No.27629, 2021 Ho Thi Oanh, Nguyen Thi Linh, Nguyen Duc Tuyen, Dang Thi Tuyet Anh, Hoang Mai Ha, Preparation of lycopene/pycnogenol nanoparticles for pharmaceutical and cosmetic applications, Vietnam Journal of Chemistry, 58(5E12), 276-281, 2020 Ho Thi Oanh, Nguyen Thanh Nhan, Hac Thi Nhung, Hoang Mai Ha, Preparation and characterization of lycopene nanoparticles, 9th National Conference of the Vietnam Organic Chemistry Association, 81-83, 2019 Ho Thi Oanh, Hoang Mai Ha, Preparation and stability study of resveratrol nanoparticles, Vietnam Journal of Chemistry, 57(6E1,2), 125129, 2019 Ho Thi Oanh, Hac Thi Nhung, Tran Thi Thanh Hop, Nguyen Duc Tuyen, Duong Thi Hai Yen, Pham Xuan Manh, Pham Duy Linh, Hoang Mai Ha, Synthesis and characterization of polylactide- based composites, The international scientific conference on Chemistry for Sustainable Development, 2018 Ho Thi Oanh, Dang Thi Tuyet Anh, Nguyen Van Tuyen, Duong Thi Hai Yen, Hac Thi Nhung, Nguyen Duc Tuyen, Pham Xuan Manh, Pham Duy Linh, Pham Thi Ha Anh, Hoang Mai Ha, Preparation and characterization of lycopene nanoparticles for pharmaceutical applications, Vietnam Journal of Chemistry, 56(4e), 27-31, 2018 Ho Thi Oanh, Hac Thi Nhung, Nguyen Duc Tuyen, Le Thi Kim Van, Trinh Hien Trung, Hoang Mai Ha, Extraction of Lycopene from Gac Fruit and Preparation of Nanolycopene, Vietnam Journal of Chemistry, 55(6), 761-766, 2017 25 ... lycopene-containing nanosystems 22 Four types of nano- products of bio-active ingredients have been prepared, including nano lycopene, nano resveratrol, lycopene /resveratrol nanosystem, and lycopene /pycnogenol nanosystem... 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... 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