Download

ORIGINAL ARTICLE

Enhancing stability and bioactivity of Chondrus ocellatus polyphenols through nanoparticle fabrication

Jinyang Shen1, Yue Zhou2*, Xue Li3, Xinxin Zhang1, Caiping Liu1, Luqian Cao1, Feiyang Zhou1, Shicheng Zhang1, Yuexia Zhu1, Kunming Qin1

1Department of Pharmacy, Jiangsu Ocean University, Lianyungang, China;

2Department of Pharmacy, Lianyungang Affiliated Hospital of Nanjing University of Chinese Medicine, Lianyungang, Jiangsu, China;

3Department of Pharmacy, Qingdao Sixth People’s Hospital, Qingdao, Shandong, China

Abstract

This article aimed to fabricate Chondrus ocellatus polyphenols (COPs)-gelatin-chitosan nanoparticles to enhance their stability and bioactivity. Different preparation conditions were tested to investigate the effects of formulation on nanoparticle fabrication. Free radical scavenging activity of COPs and their nanoparticles were compared. The consequences revealed that optimal preparation was obtained with a chitosan (CS) concentration of 0.5 mg/mL, gelatin (Gel) concentration of 1.0 mg/mL, COPs concentration of 5.0 mg/mL, and Gel-CS-COPs mass ratio of 2:1:1. The resultant nanoparticles had the particle size of 39.79 ± 5.15 nm and encapsulation efficiency of 60.95 % ± 1.86 %. The COPs-Gel-CS nanoparticles were distributed uniformly, and no obvious aggregation was observed by transmission electron microscopy. Nanoencapsulation of the COPs significantly improved their antioxidative stability. This study provided a potential formulation for the application of Chondrus ocellatus polyphenols in antioxidant activities.

Key words: antioxidative stability, characterization, Chondrus ocellatus, nanoparticle, polyphenols, preparation

*Corresponding Author: Yue Zhou, Department of Pharmacy, Lianyungang Affiliated Hospital of Nanjing University of Chinese Medicine, No. 160, Chaoyang Middle Road, Haizhou District, Lianyungang, China. Email: [email protected]

Academic Editor: Prof. Valeria Sileoni - Universitas Mercatorum, Italy.

Received: 26 June 2024; Accepted: 6 August 2024; Published: 11 November 2024

DOI: 10.15586/ijfs.v36i4.2674

© 2025 Codon Publications
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0). License (http://creativecommons.org/licenses/by-nc-sa/4.0/)

Introduction

A distinctive place is reserved for phenolic compounds in marine algae, as these algae have been recognized as a rich source of biologically active phenolic compounds (Thomas and Kim, 2011). Owing to their broad spectrum of antioxidant activities, these compounds have been recognized as having protective effects against many diseases, including cardiovascular diseases, diabetes, cancer, atherosclerosis, aging, and other degenerative diseases (Fernando et al., 2016; Jayawardhana et al., 2021). However, phenolic compounds are sensitive to oxidation and photolysis during utilization and storage and have low boiling points, volatility, and poor stability. Therefore, increasing the stability of phenolic compounds is expected to enhance their biological activities significantly (Carbonaro et al., 2001; Chen et al., 2023; Zou et al., 2012).

Nanoparticles were first reported in the 1980s and defined as particulate dispersions or solid particles with a size range of 1–1000 nm (Mohanraj and Chen, 2006). Nanoparticles are not only small in size, and easy to be taken up by cells, but also have good biocompatibility and targeting properties (Stark et al., 2015). Therefore, drugs or other bioactive compounds encapsulated in nanoparticles exhibit higher stability, bioavailability, and bioactivity (Guo et al., 2021; Roger et al., 2010). Gelatin (Gel) is a natural protein-based biopolymer with relatively low antigenicity. Its biodegradability, biocompatibility, chemical modification potential, and cross-linking possibilities make Gel nanoparticles a promising drug delivery carrier system (Young et al., 2005). Chitosan (CS) is a nontoxic, safe, and nonantigenic natural cationic polysaccharide that has been widely used as a carrier for nanoparticles (Liang et al., 2017; Pillai et al., 2009). Nanoparticles have been attempted as drug delivery systems for some phenolic compounds. In one study, the encapsulation of cocoa procyanidins in Gel-CS nanoparticles significantly improved their stability (Zou et al., 2012). Encapsulation of catechins in CS nanoparticles significantly enhanced their intestinal absorption by 1.5 fold (Dube et al., 2010).

Chondrus ocellatus is a kind of red algae with clusters, and it has been reported as a potential source of antioxidant, antitumor, antiviral, anticoagulant, and immunomodulatory active substances (Zhou et al., 2005). In our previous studies, polyphenols were extracted from C. ocellatus and found to have antioxidant and inhibitory activities of α-amylase and α-glycosidase (Zhu et al., 2022). However, a suitable drug delivery system still needs to be developed for further development and utilization of C. ocellatus polyphenols (COPs). The COPs-Gel-CS nanoparticle system was planned to improve the stability of COPs, expand the application scope, and provide a reference for the development and utilization of other natural polyphenols. Thus, the objective of this study was to fabricate COPs-Gel-CS nanoparticles and to evaluate their advantages.

Material and Methods

Materials

Chondrus ocellatus was collected from the seaside of Xiapu, Fujian, China. Gel (250 bloom) and CS (deacetylation degree of 95 %, viscosity of 100–200 mpa.s) were purchased from Macklin Biochemical Technology Co., Ltd. All other chemicals and solvents were of analytical grade and were obtained from Macklin Biochemical Technology Co., Ltd unless otherwise indicated.

Extraction of COPs

COPs were extracted according to the procedure from Zhu et al. (2022). C. ocellatus was washed with running tap water for several minutes and fully dried. The dried C. ocellatus was pulverized by a laboratory blender for several minutes and sieved to produce fine powder for further use. The thin powder was dissolved in 61%(v/v) hydroethanolic solution at the ratio of material to liquid of 1:25 (w/v), extracted by ultrasonic at 60°C for 41 min, and the extract was dried in a vacuum to obtain polyphenol solid.

Preparation of the COPs-Gel-CS nanoparticles

The COPs-Gel-CS nanoparticles were prepared according to the methods reported by Sun et al. (2020) with some modifications. Previous studies showed that the solubility and fluidity of CS are good at pH 5.4 (Zhang et al., 2014). The specific preparation method was determined by single factor experiment and orthogonal experiment. CS was dissolved in 0.5 % acetate solution (v/v) to produce a concentration of 0.5 mg/mL. Gel solution (1.0 mg/mL) was obtained in distilled water by stirring at 40°C. COPs were dissolved in 40% ethanol solution (v/v) at a concentration of 5.0 mg/mL. Subsequently, the COPs solution was completely dissolved into the CS solution. Then, the mixture was added to the gel solution at a slow rate. The gel-CS-COPs mass ratio was 2:1:1. The mixture was magnetically stirred at room temperature for 10 min at 550 rpm/min, adjusted pH to 5.4 with 0.2 M NaOH, and finally mixed for another 30 min to obtain a suspension of nanoparticles.

Characterization and antioxidation of the COPs-Gel-CS nanoparticles

Particle size analysis

Mean particle size and polydispersion index (PDI) of the COPs-Gel-CS nanoparticles was performed by Zetasizer Nano-ZS (Malvern Instruments, UK). An appropriate amount of COPs-Gel-CS nanoparticles was pipetted out into the measuring dish, with the height of the sample controlled within 1–1.5 cm. Measurements were made according to the computer program. Samples were measured in triplicate to calculate the average particle size.

Encapsulation efficiency

For the prepared nanoparticles, the encapsulation efficiency was measured using the supernatant of the suspension obtained by centrifugation according to references (Ma et al., 2020). The suspension of the COPs-Gel-CS nanoparticles was separated by centrifugation at 12,000 rpm/min for 30 min at 4°C. The mass of COPs used for nanoparticle synthesis and the mass of COPs in the supernatant were measured using UV-Vis spectrophotometry at a detection wavelength of 760 nm. The following formula was used to calculate the encapsulation efficiency:

Encapsulation efficiency %=MtMnMt×100

where Mt was the mass of COPs used for nanoparticle synthesis. Mn was the mass of COPs in the supernatant.

Transmission electron microscopy (TEM) analysis

The morphology of nanoparticles was analyzed using TEM according to the methods reported by Chanphai and Tajmir-Riahi (2018) with some modifications. The appropriately prepared samples were placed on carbon-coated copper grids after dilution. After standing for a while, the samples were negatively dyed with phosphotungstic acid for several minutes and dried naturally. Then, the morphology and distribution of nanoparticles were imaged using TEM (Model JEM-1400 plus, JEOL Ltd, Tokyo, Japan).

Antioxidative stability

The absorbance of the nanoparticles was measured using the 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) di-ammonium salt (ABTS) method to determine their antioxidant properties according to the method reported by Sun et al. with some modifications (Sun et al., 2021). ABTS kit (BC4770, Solarbio) was used according to the protocol of the kit. In this kit, the degree of decrease in absorbance was measured to reflect the ability of the sample to scavenge ABTS-free radicals.

For the stability study, 5 mg/mL of COPs and COPs-Gel-CS nanoparticles with an equivalent quantity of COPs were stored at 4°C and 37°C, respectively, to determine the change of free radical scavenging for a set period (1, 2, 3, 4, and 5 h).

Statistical analysis

Samples were analyzed in triplicate, and the average values were used. All the experimental data were expressed as mean ± standard deviation. Statistical analyses were performed using Student’s t-test, and P < 0.05 was considered to be significantly different (Lee et al., 2019).

Results and Discussion

Preparation of the COPs-Gel-CS nanoparticles

Effect of gel and CS concentration on encapsulation efficiency

COPs nanoparticles were prepared by following the coacervation method. The rupture of hydrogen bonds between water and Gel molecules brought the complementary charged segments of gelatin closer (coacervates) (Lee et al., 2011). Consequently, negatively charged gelatin and positively charged chitosan self-assembled and encapsulated the polyphenols to form COPs nanoparticles. Turbidity and particle size changes of the solution system prepared by different mass ratios of CS and Gel are shown in Figure 1.

Figure 1. Effect of Gel-CS mass ratio on turbidity and particle size. The numbers in the abscissa denote the gelatin to chitosan ratio, and 1–9 represent gelatin: chitosan ratios of 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, and 9: 1, respectively.

The results showed that when the mass ratio was 1:1, the content of protonated CS in the system was low. Its hydrophilicity was not affected after mixing with Gel, and it could be evenly dispersed in water. With the increase of the molecular weight ratio of Gel, the complex structure was compact, and the particle size reached the minimum at 2:1. A pale blue opalescence with the gender effect can be seen from the surface. With the increase in gel mass ratio, more free molecules were generated, which affected the stable crosslinking of molecules. Blue opalescence disappeared and white flocculent condensates precipitated.

Investigation of factors affecting particle size

We investigated the effects of prescription factors (CS concentration, Gel concentration, COPs concentration, and CS-COPs mass ratio) on the particle size of COPs-Gel-CS nanoparticles. The results are shown in Figure 2. The results showed that nanoparticles with larger particle sizes could be formed with a higher concentration of nanocarrier and nanodrug, and the mass ratio of carrier to the drug was close to equal. The study also examined the impact of manufacturing processes (preparation time and rotation speed) on nanoparticle size, as shown in Figures 2E and 2F. Both time and rotation speed were found to significantly affect particle size. A preparation time of 20 min and a stirring speed of 550 r/min were chosen accordingly.

Figure 2. Effect of each factor on particle size.

To further reflect the effects of prescription factors on the particle size, the results of the orthogonal test are shown in Tables 13. The order of influence was A (CS concentration) > B (Gel concentration) > C (COPs concentration) > D (CS-COPs mass ratio). The optimal conditions were A2B1C2D2. CS, Gel, and COPs concentrations and CS-COPs mass ratio were 0.5 mg/mL, 1.0 mg mL, 5.0 mg/mL, and 1:1, respectively. The verification results showed that the particle size of COPs-Gel-CS nanoparticles was 39.79 ± 5.15 nm.

Table 1. Orthogonal test program.

Symbol Independent variables levels
1 2 3
A CS concentration (mg/mL) 0.25 0.5 1.0
B Gel concentration (mg/mL) 1.0 2.0 3.0
C COPs concentration (mg/mL) 4.0 5.0 6.0
D CS-COPs mass ratio (m/m) 2:1 1:1 1:2

Table 2. Orthogonal test results.

No. CS concentration
(mg/mL)
Gel concentration
(mg/mL)
COPs concentration
(mg/mL)
CS-COPs mass ratio (m/m) Size
(nm)
1 0.25 1.0 4.0 2:1 74.59
2 0.25 2.0 5.0 1:1 67.84
3 0.25 3.0 6.0 1:2 68.70
4 0.5 1.0 5.0 1:2 39.72
5 0.5 2.0 6.0 2:1 56.39
6 0.5 3.0 4.0 1:1 57.20
7 1.0 1.0 6.0 1:1 71.70
8 1.0 2.0 4.0 1:2 92.20
9 1.0 3.0 5.0 2:1 87.82
Mean 1 70.377 62.003 74.663 72.933
Mean 2 51.103 72.143 65.127 65.580
Mean 3 83.907 71.240 65.597 66.873
Range 32.804 10.140 9.536 7.353
Sequence A>B>C>D
Optimal Combination A2B1C2D2

Table 3. Orthogonal test variance analysis table.

Source of variance Sums of squared deviations Degree of freedom Mean square F P Significance
A 1630.5810 2 543.52700 17.63404 0.020762 *
B 188.9516 2 62.98387 2.043431 0.286092 No significant difference
C 173.3734 2 57.79112 1.874959 0.309286 No significant difference
D 92.46782 2 30.82261 1.000000 0.500000 No significant difference

*P < 0.05.

Particle size, Polymer dispersity index (PDI), and encapsulation efficiency

As shown in Figures 3 and 4, under the optimal preparation process, the particle size and zeta potential of Gel-Cs-COPs nanoparticles were 39.79 nm and 32.9 ± 7.08 mV, respectively.

Figure 3. Size distribution of Gel-Cs-COPs nanoparticles.

Figure 4. Zeta potential distribution of Gel-Cs-COPs nanoparticles.

PDI was 0.236, which indicated that the nanoparticle has good dispersion and the system was stable (Gaumet et al., 2008). The encapsulation efficiency of COPs in the Gel-Cs-COPs nanoparticles was 60.95 ± 1.86%. Encapsulation efficiency is an important index of plant polyphenol nanoparticles. Similar to Gel-Cs-COPs nanoparticles in this study, the encapsulation efficiency of litchi polyphenol-loaded chitosan nanoparticles reported by Cheng et al. (2023) is 45.9%, and the kaempferol-loaded silk fibroin nanoparticles reported by Yang et al. (2022) is 53.8%, which is lower than the encapsulation efficiency prepared in our investigation, demonstrating the benefits of chitosan–gelatin composite capsules in this study. However, the encapsulation efficiency of sodium alginate composite cross-linked corn starch tea polyphenols nanoparticles prepared by Bu et al. (2023) can reach 78.4%, which is higher than the encapsulation efficiency of the nanoparticles in this study, indicating that sodium alginate-cross-linked corn starch composite capsule material may have a better encapsulation effect.

TEM analysis

As shown in Figure 5, the formation of nanoparticles was confirmed by the TEM images. It should be noted that when preparing TEM samples, the shell structure of phosphotungstic acid was loose and the core layer was dense, which made it difficult for phosphotungstic acid to penetrate the core layer during the red staining process, so the color of the core layer was relatively light. It could be seen that the Gel-Cs-COPs nanoparticles were spherical with uniform distribution, and no obvious aggregation was observed. The diameter of most particles was less than 50 nm. Compared to other nanoparticles, such as the biosynthesized silver nanoparticles (AgNPs) with the aid of a combination of chitosan and seaweed-derived polyphenols reported by Rezazadeh et al. (2020), our Gel-Cs-COPs nanoparticles had similar particle size, uniform distribution, and composite nanoparticle standard.

Figure 5. TEM micrograph of COPs-Gel-CS nanoparticles. The two images were observed at different sites of the nanoparticle solution on a slide at the same magnification.

Antioxidative stability

The free radical scavenging activity of free COPs and Gel-Cs-COPs nanoparticles was studied by 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) di-ammonium salt, ABTS assay (Figure 6). Under the same condition, compared with free COPs, COPs coated with gelatin–chitosan were more stable. Even at 5 h, COPs-Gel-Cs nanoparticles still had higher stability and stronger free radical scavenging activity. Meanwhile, the free radical scavenging activity of those stored at 4°C was higher than that stored at 37°C. Due to the higher temperature, COPs were more susceptible to oxidative degradation by oxidation reactions. The Gel-Cs-COPs nanoparticles maintained high scavenging activity at different temperatures and times, while the scavenging activity of COPs decreased significantly with the increase of time. The results confirmed that the preparation of COPs into nanoparticles increased their antioxidant stability by avoiding their volatile and poor stability properties.

Figure 6. Free radical scavenging activity of free COPs and Gel-Cs-COPs nanoparticles as measured by ABTS assay.

Discussion

Dosage forms of plant polyphenols have been studied intensively. Due to the instability of polyphenol properties, raw materials will be consumed under the conventional dosage form preparation conditions (Li et al., 2020; Yang et al., 2020). Similarly, the properties of C. ocellatus polyphenols are unstable and prone to oxidation and photolysis. To improve the utilization rate of polyphenols, new dosage forms such as microcapsules and clathrates were gradually used by researchers to improve the physical properties of plant polyphenols. For example, Alizadeh and Nazari (2022) prepared thymol (TH)/β-cyclodextrin (β-CD)-inclusion complex (ICs) clathrate using β-CD and CS as nanocarriers to reduce its oxidation and photolysis and effectively improved the storage and utilization of thymol. In addition to β-CD and CS, sodium alginate, pectin (Sun et al., 2022), gelatin, and so on were commonly used as carriers for the preparation of microcapsules. The Camellia chrysantha polyphenols nanoparticles prepared by Luong et al. (2021) used sodium alginate and CS as carriers to improve the solubility of tea polyphenols. In our study, to reduce the loss of C. ocellatus polyphenols due to their oxidation and photolysis, the nanoparticles with antioxidant and photolytic activity were prepared using chitosan and gelatin as carriers. Mathew and Arumainathan (2022) demonstrated that the microcapsules prepared by chitosan had pretty biological properties, but chitosan had pH-responsive drug release, which needed to be effectively released under acidic conditions. However, the compounding of chitosan and gelatin facilitated continuous release in a neutral medium, and the chitosan–gelatin microcapsules had plummy solubility and content release properties. Therefore, the use of polyphenols was enhanced to a certain extent by using chitosan and gelatin to prepare composite microcapsule materials, which could alleviate the disadvantages caused by the unstable properties of C. ocellatus polyphenols. (Lv et al., 2018). Menezes et al. (2016) prepared βCD carvacrol microcapsules using physical mixture (PM), paste completion (PC), and slurry completion (SC) methods. The encapsulate efficiency of the microcapsules prepared by PM and PC methods was 2.96 ± 0.015 and 34.30 ± 0.24%, respectively, indicating a low entrapment efficiency. The encapsulation efficiency of the microcapsules prepared by SC method was improved to 71.68 ± 0.062%. However, the process of preparing the composite dry powder requires additional experimental steps and is relatively time-consuming. Furthermore, compared with a single carrier, composite carriers have drawn more and more attention due to the stable interaction between them without the need to add crosslinkers as well as their good drug-loading properties (Sethi et al., 2022). In our study, the positive charge of chitosan after dissolution could form a stable interaction with the negative charge of gelatin. After adding C. ocellatus polyphenols, gelatin, and chitosan could encapsulate the polyphenols to form the C. ocellatus nanoparticles in the process of synthesis and formation of nanoparticles due to electrostatic interaction. The components of the polyphenols were relatively complex and other components might influence the formation of the nanoparticles to a certain extent. The encapsulation efficiency was 60.95 ± 1.86%, and the preparation process was simple physical mixing. The formed nanoparticles were clear in appearance. The particles were regular and round, and the antioxidant performance of the seaweed polyphenol was improved (Tao et al., 2022).

In this study, the gelatin–chitosan complex was prepared by complex coacervation. Gelatin was a protein and molecular chains contained -NH2 and -COOH and their corresponding dissociation groups -NH3+ and -COO-, but the amount of ions containing-NH3+ and -COO- was affected by the pH of the medium. When pH was lower than the isoelectric point of gelatin, the number of -NH3+ was more than that of -COO-, and the solution had a positive charge. When the pH of the solution was higher than that of the gelatin isoelectric point, -NH3+ was less than that of -COO-, and the solution became negatively charged (Sethi et al., 2022). The negative charge of the gelatin solution was the highest at pH 5. Chitosan was a positively charged polyelectrolyte in a solution with strong adsorption. In this study, chitosan dissolved in 0.5% acetic acid formed a positively charged cationic group in the solution. The C. ocellatus polyphenols alcohol solution was injected into the chitosan solution in advance, and the composite solution was added into the gelatin solution according to proportion. After pH was adjusted, the chitosan and the gelatin were mutually aggregated due to electrostatic interaction between charges, and the C. ocellatus polyphenols were encapsulated to form nanoparticles. The microcapsule formed a closed protective layer which was not reacted with the closed protective layer and was stably combined with the polyphenol outer layer, so that the gel-CS microcapsule could contact with oxygen and light, thus reducing its oxidation and photolysis.

Conclusion

In conclusion, we developed an optimal preparation method for C. ocellatus polyphenols nanoparticles. Under these conditions, the encapsulation efficiency could reach 60.95 ± 1.86%. TEM analysis demonstrated the formation of uniform spherical nanoparticles. The antioxidant experiments showed that the nanoparticles could effectively alleviate the problem of polyphenol oxidation and enhance its stability. This study provided ideas for further application of polyphenols. However, this study mainly focused on the optimization of the preparation conditions of nanoparticles and the determination of antioxidant activity in vitro. It is also necessary to further study the performance of polyphenol nanoparticles prepared by this technology in the human body.

Acknowledgements

This project was funded by the Jiangsu Province Chinese Medicine Science and Technology Development Programme Project Face-up Project (MS2023178), the Lianyungang TCM science and technology development plan project (ZD202203), the Key Project of Lianyungang Chinese Medicine Science and Technology Project (LZYZD202403) and Postgraduate Research & Practice Innovation Program of Jiangsu Province.

Funding

This project was funded by the Jiangsu Province Chinese Medicine Science and Technology Development Programme Project Face-up Project (MS2023178), the Lianyungang TCM science and technology development plan project (ZD202203) and Postgraduate Research & Practice Innovation Program of Jiangsu Province.

Conflict of Interest

The authors have declared no conflicts of interest for this article.

REFERENCES

Alizadeh, N., and Nazari, F., 2022. Thymol essential oil/β-cyclodextrin inclusion complex into chitosan nanoparticles: Improvement of thymol properties in vitro studies. Journal of Molecular Liquids. 346(2): 118250. 10.1016/j.molliq.2021.118250

Bu, Q., Chen, Y., Ding, Y., Zhang, K.X., Li, Y.C., You, X.Y., et al., 2023. Preparation and characterization of tea polyphenol composite microspheres encapsulated using sodium alginate and crosslinked starch. LWT. 184: 114888. 10.1016/j.lwt.2023.114888

Carbonaro, M., Grant, G., and Pusztai, A., 2001. Evaluation of polyphenol bioavailability in rat small intestine. European Journal of Nutrition. 40(2): 84–90. 10.1007/s003940170020

Chanphai, P., and Tajmir-Riahi, H.A., 2018. Binding analysis of antioxidant polyphenols with PAMAM nanoparticles. Journal of Biomolecular Structure and Dynamics. 36(13): 3487–3495. 10.1080/07391102.2017.1391124

Chen, H., Lin, S., Wu, J., Xu, Y., Cai, X., and Wang, S., 2023. The structure, antioxidant activity, and stability of fish gelatin/chitooligosaccharide nanoparticles loaded with apple polyphenols. Journal of Science of Food and Agriculture. 103(8): 4211–4220. 10.1002/jsfa.12455

Cheng, X., Zou, Q., Zhang, H., Zhu, J., Hasan, M., Dong, F., et al., 2023. Effects of a chitosan nanoparticles encapsulation on the properties of litchi polyphenols. Food Science and Biotechnology. 32(13): 1861–1871. 10.1007/s10068-023-01303-3

Dube, A., Nicolazzo, J.A., and Larson, I., 2010. Chitosan nanoparticles enhance the intestinal absorption of the green tea catechins (+)-catechin and (−)-epigallocatechin gallate. European Journal of Pharmaceutical Sciences. 41(2): 219–225. 10.1016/j.ijpharm.2021.121382

Fernando, I.P.S., Kim, M., Son, K.T., Jeong, Y., and Jeon, Y.J., 2016. Antioxidant activity of marine algal polyphenolic compounds: A mechanistic approach. Journal of Medicinal Food. 19(7): 615–628. 10.1089/jmf.2016.3706

Gaumet, M., Vargas, A., Gurny, R., and Delie, F., 2008. Nanoparticles for drug delivery: The need for precision in reporting particle size parameters. European Journal of Pharmaceutics and Biopharmaceutics. 69(1): 1–9. 10.1016/j.ejpb.2007.08.001

Guo, Y., Sun, Q., Wu, F.-G., Dai, Y., and Chen, X., 2021. Polyphenol-containing nanoparticles: Synthesis, properties, and therapeutic delivery. Advanced Materials. 33(22): 2007356. 10.1002/adma.202007356

Jayawardhana, H.H., Jayawardena, T.U., Sanjeewa, K.K.A., Liyanage, N.M., Nagahawatta, D.P., Lee, H.G., et al., 2023. Marine algal polyphenols as skin protective agents: Current status and future prospectives. Marine Drugs. 21(5): 285. 10.3390/md21050285

Lee, E.J., Khan, S.A., and Lim, K.H., 2011. Gelatin nanoparticle preparation by nanoprecipitation. Journal of Biomaterial Science, Polymer Edition. 22(4): 753–771. 10.1163/092050610X492093

Lee, I.C., Lee, J.S., Lee, J.H., Kim, Y., and So, W.Y., 2019. Anti-oxidative and anti-inflammatory activity of Kenya Grade AA green coffee bean extracts. Iranian Journal of Public Health. 48(11): 2025–2034. 10.18502/ijph.v48i11.3521

Li, D., Zhu, M., Liu, X., Wang, Y., and Cheng, J., 2020. Insight into the effect of microcapsule technology on the processing stability of mulberry polyphenols. LWT. 126: 109144. 10.1016/j.lwt.2020.109144

Liang, J., Yan, H., Puligundla, P., Gao, X., Zhou, Y., and Wan, X., 2017. Applications of chitosan nanoparticles to enhance absorption and bioavailability of tea polyphenols: A review. Food Hydrocolloids. 69: 286–292. 10.1016/j.foodhyd.2017.01.041

Luong, P.H., Nguyen, T.C., and Pham, T.D., 2021. Preparation and assessment of some characteristics of nanoparticles based on sodium alginate, chitosan, and Camellia chrysantha polyphenols. International Journal of Polymer Science. 2021: 5581177. 10.1080/07391102.2017.1391124

Lv, Y., He, H., Qi, J., Lu, Y., Zhao, W., Dong, W., et al., 2018. Visual validation of the measurement of entrapment efficiency of drug nanocarriers. International Journal of Pharmaceutics. 547(1): 395–403. 10.1016/j.ijpharm.2018.06.025

Ma, Y., Li, S., Ji, T., Wu, W., Sameen, D.E., Ahmed, S., et al., 2020. Development and optimization of dynamic gelatin/chitosan nanoparticles incorporated with blueberry anthocyanins for milk freshness monitoring. Carbohydrate Polymers. 247: 116738. 10.1016/j.carbpol.2020.116738

Mathew, S.A., and Arumainathan, S., 2022. Crosslinked chitosan–gelatin biocompatible nanocomposite as a neuro drug carrier. ACS Omega. 7(22): 18732–18744. 10.1021/acsomega.2c01443

Menezes, P.P., Serafini, M.R., de Carvalho, Y., Santana, D.V.S., Lima, B.S., Quintans-Júnior, L.J., et al., 2016. Kinetic and physical-chemical study of the inclusion complex of β-cyclodextrin containing carvacrol. Journal of Molecular Structure. 1125: 323–330. 10.1016/j.molstruc.2016.06.062

Mohanraj, V.J., and Chen, Y., 2006. Nanoparticles-a review. Tropical Journal of Pharmaceutical Research. 5(1): 561–573. 10.4314/tjpr.v5i1.14634

Pillai, C.K.S., Paul, W., and Sharma, C.P., 2009. Chitin and chitosan polymers: Chemistry, solubility and fiber formation. Progress in Polymer Science. 34(7): 641–678. 10.1016/j.progpolymsci.2009.04.001

Rezazadeh, N.H., Buazar, F., and Matroodi, S., 2020. Synergistic effects of combinatorial chitosan and polyphenol biomolecules on enhanced antibacterial activity of biofunctionalized silver nanoparticles. Scientific Reports. 10(1): 19615. 10.1038/s41598-020-76726-7

Roger, E., Lagarce, F., Garcion, E., and Benoit, J.P., 2010. Biopharmaceutical parameters to consider in order to alter the fate of nanocarriers after oral delivery. Nanomedicine (London, England). 5(2): 287–306. 10.2217/nnm.09.110

Sethi, S., Medha, Kaith BS., 2022. A review on chitosan-gelatin nanocomposites: Synthesis, characterization and biomedical applications. Reactive and Functional Polymers. 179: 105362. 10.1016/j.reactfunctpolym.2022.105362

Sethi S, Medha, K., and Singh, G., 2022. Fluorescent hydrogel of chitosan and gelatin cross-linked with maleic acid for optical detection of heavy metals. Journal of Applied Polymer Science. 13(15): 5581177. 10.1002/app.51941

Stark, W.J., Stoessel, P.R., Wohlleben, W., and Haffner, A., 2015. Industrial applications of nanoparticles. Chemical Society Reviews. 44(16): 5793–5805. 10.1039/c4cs00362d

Sun, C., Cao, J., Wang, Y., Huang, L., Chen, J., Wu, J., et al., 2022. Preparation and characterization of pectin-based edible coating agent encapsulating carvacrol/HPβCD inclusion complex for inhibiting fungi. Food Hydrocolloids. 125: 107374. 10.1016/j.foodhyd.2021.107374

Sun, L., Lu, B., Liu, Y., Wang, Q., and Zhao, C., 2021. Synthesis, characterization and antioxidant activity of quercetin derivatives. Synthetic Communications. 51(19): 2944–2953. 10.1080/00397911.2021.1942059

Sun, M., Xie, Q., Cai, X., Liu, Z., Wang, Y., and Dong, X., et al., 2020. Preparation and characterization of epigallocatechin gallate, ascorbic acid, gelatin, chitosan nanoparticles and their beneficial effect on wound healing of diabetic mice. International Journal of Biological Macromolecules. 148: 777–784. 10.1016/j.ijbiomac.2020.01.198

Tao, X., Shi, H., Cao, A., Cai, L., 2022. Understanding of physicochemical properties and antioxidant activity of ovalbumin–sodium alginate composite nanoparticle-encapsulated kaempferol/tannin acid. RSC Advances. 12(28): 18115–18126. 10.1039/D2RA02708A

Thomas, N.V., and Kim, S.K., 2011. Pharmacological applications of polyphenolic derivatives from marine brown algae. Environmental Toxicology and Pharmacology. 32(3): 325–335. 10.1016/j.etap.2011.09.004

Yang, B., Dong, Y., Wang, F., and Zhang, Y., 2020. Nanoformulations to enhance the bioavailability and physiological functions of polyphenols. Molecules. 25(20): 4613. 10.3390/molecules25204613

Yang, W., Xie, D., Liang, Y., Chen, N., Xiao, B., Duan, L., et al., 2022. Multi-responsive fibroin-based nanoparticles enhance anti-inflammatory activity of kaempferol. Journal of Drug Delivery Science and Technology. 68: 103025. 10.1016/j.jddst.2021.103025

Young, S., Wong, M., Tabata, Y., and Mikos, A.G., 2005. Gelatin as a delivery vehicle for the controlled release of bioactive molecules. Journal of Controlled Release. 109(1): 256–274. 10.1016/j.jconrel.2005.09.023

Zhang, Y., Meng, C., Chang, J., Sha, S., Chen, H., and Lu, S., 2014. Preparation and characterization of a self-assembled tea polyphenol-gelatin-chitosan nanoparticles. Journal of China Pharmaceutical University. 68(14): 1399–1403. 10.1631/jzus.B1000073

Zhou, G., Xin, H., Sheng, W., Sun, Y., Li, Z., and Xu, Z., 2005. In vivo growth-inhibition of S180 tumor by mixture of 5-Fu and low molecular λ-carrageenan from Chondrus ocellatus. Pharmacological Research. 51(2): 153–157. 10.1016/j.phrs.2004.07.003

Zhu, Y., Chen, W., Kong, L., Zhou, B., Hua, Y., Han, Y., et al., 2022. Optimum conditions of ultrasound-assisted extraction and pharmacological activity study for phenolic compounds of the alga Chondrus ocellatus. Journal of Food Processing and Preservation. 46(3): e16400. 10.1111/jfpp.16400

Zou, T., Percival, S.S., Cheng, Q., Li, Z., Rowe, C.A., and Gu, L., 2012. Preparation, characterization, and induction of cell apoptosis of cocoa procyanidins–gelatin–chitosan nanoparticles. European Journal of Pharmaceutics and Biopharmaceutics. 82(1): 36–42. 10.1016/j.ejpb.2012.05.006