Creating burdock polysaccharide-oleanolic acid-ursolic acid nanoparticles to deliver enhanced anti-inflammatory effects: fabrication,structural characterization and property evaluation

Shnshn Zhu,Zhihng Qiu,Xugung Qio,Geoffrey I.N.Wterhouse,Wenqing Zhu,Wenting Zho,Qiuxi He,Zhenji Zheng,*

a Key Laboratory of Food Processing Technology and Quality Control of Shandong Higher Education Institutes,College of Food Science and Engineering,Shandong Agricultural University,Tai’an 271018,China.

b The School of Chemical Sciences,The University of Auckland,Auckland 1025,New Zealand.

c Institute of Agri-food Processing and Nutrition,Vegetable Research Center,Beijing Academy of Agriculture and Forestry Sciences,Beijing 100097,China.

d Biology Institute,Qilu University of Technology (Shandong Academy of Sciences),Jinan 250103,China

Keywords:Encapsulation Structural features Particle size Zeta potential Thermodynamic properties In vivo verification

ABSTRACT This study explored the potential of polysaccharides from Actium lappa (ALPs) as natural wall materials for producing ALP-based nanoparticles to deliver poorly water-soluble oleanolic acid (OA) and ursolic acid(UA).Encapsulating OA+UA with ALPs (ALP:OA+UA,50:1;OA:UA,1:1) changed the crystalline nature to a more amorphous state through hydrogen bonding and involving O–H/C–O/O–C–O groups.ALP-OA/UA nanoparticles had a particle size and zeta potential (in water) of 199.1 nm/-7.15 mV,with a narrow unimodal size distribution,and excellent pH,salt solution,temperature and storage stability.Compared with ALPs,ALPOA/UA nanoparticles showed enhanced anti-inflammatory activity (especially at a dose of 100 μg/mL) in a CuSO4-induced zebrafish inflammation model via down-regulating the NF-κB signalling pathway and gene expression of associated transcription factors and cytokines (TNF-α,IL-1β and IL-8).Therefore,ALP-based nanoparticles are natural and anti-inf lammatory carriers for hydrophobic bioactive molecules.

1.Introduction

Oleanolic acid (OA,3β-hydroxy-urs-12-en-28-oic-acid)and ursolic acid (UA,3β-hydroxy-olea-12-en-28-oic-acid) are natural pentacyclic triterpenoid carboxylic compounds abundant in medicinal herbs and fruits [1].As triterpine isomers,they have similar chemical structures and pharmacological activities,including anti-inflammatory,hepatoprotective,antioxidant,antitumor and immunoregulatory effects [2].In recent years,OA and UA have been used to develop novel functional foods and drugs due to their availability,low toxicity and high efficacy,especially their antiinflammatory effects [3].UA was found to modulate the activation of nuclear factor-κB (NF-κB) in human intestinal epithelial cells and peritoneal macrophages stimulated by tumor necrosis factor-α(TNF-α) and lipopolysaccharide (LPS) from interleukin-10(IL-10)-deficient (IL-10?/?) mice via down-regulating proinflammatory cytokines and suppressing inhibitor of NF-κB (IκBα)phosphorylation/degradation and NF-κB DNA binding activity [4].In the same study,UA also exhibited the ability to ameliorate intestinal inflammation in dextran sulphate sodium (DSS)-induced acute colitis mice and IL-10?/?piroxicam-induced chronic colitis mice [4].The administration of OA was reported to alleviate spinal cord injury through reducing the phosphorylation of p38,c-Jun-NH 2 terminal kinase (JNK),IκB kinase α (IKKα),IκBα and NF-κB [5].Furthermore,abnormal colon shortening,macroscopic scores and myeloperoxidase activity in mice with DSS-induced acute colitis were effectively alleviated after the intervention of OA,which was mainly attributed to the inhibition of Th17 cell differentiation,downregulation of NF-κB and mitogen-activated protein kinases (MAPKs)activation and TNF-α,IL-1β and IL-17 (including its transcription factor RORγt) expression,enhancement of Treg cell differentiation and tight junction protein expression in the colon,as well as upregulation of IL-10 expression (including its transcription factor Foxp3) [6].However,most published studies have focused on the anti-inflammatory effects of OA alone or UA alone,with only a few reporting the combined effects of OA and UA.Since OA and UA usually coexist in natural plants,it is of interest to examine whether synergy exists in their anti-inflammatory effects.

Both OA and UA have poor aqueous solubility,which greatly limits their bioavailability and application in the food and pharmaceutical industries.Nanonization is an efficient approach for increasing solubility and bioavailability [7].Encapsulation technologies offer solutions to overcome the instability and insolubility issues of bioactive substances [8,9].Nanoparticle-based carriers are promising delivery systems for bioactive compounds and offer advantages in terms of simple and low-cost synthesis process,increased aqueous solubility,enhanced enteric epithelial cell absorption,high biocompatibility and nontoxicity [10].Ingestion of large amounts of surfactants may cause potential damage to the human body,therefore,natural biopolymer-based delivery systems produced in the absence of surfactants are preferred [11].As a class of nutrients and polymeric molecules abundant in nature,polysaccharides have been widely used as wall materials for the encapsulation of bioactive substances [8,9,12,13].Burdock (Actium lappa) is known to contain large amounts of non-starch polysaccharides (prebiotic fibers) [14].Our research team found that the main polysaccharides fromArctium lappa(ALPs) had a linear chain of (2→1)-linkedβ-D-fructofuranosyl backbone attached to a terminal (2→1)-linkedα-D-glucopyranosyl group and exhibited excellent antioxidant activities [15].Inulin-type fructans represent a class of linear fructan-type plant polysaccharides composed of fructosyl units [β-(2→1) linkage]and usually terminated by aD-glucopyranosyl residue [α-(2→1) linkage],which makes it indigestible in the human small intestine and fermented by gut microflora in large intestine [16].Therefore,ALPs were considered to be an inulin-type polysaccharide with multiple health benefits.A fraction of these polysaccharides fromA.lappawas reported to significantly ameliorate DSS-induced colitis in mice [17].Therefore,using such fiber polysaccharides as wall materials for encapsulating bioactive compounds not only improves the solubility and stability of bioactive compounds,but also increases the consumption of these antioxidant and anti-inflammatory dietary fibers.

The present study firstly investigated the suitability of ALPs as natural wall materials for ALP-based nanoparticles to deliver OA and UA,then examined the advantages of such ALP-OA/UA nanoparticles (including the combined anti-inflammatory effects of ALPs,OA and UA).Before the preparation of ALP-OA/UA nanoparticles,screening the optimal ratio between OA and UA was performed in a CuSO4-induced zebrafish inflammation model.This model is commonly used for the development of anti-inflammatory agents,as zebrafish immune system has almost all human immune system cells and CuSO4can induce neuronal damage in the lateral line system of zebrafish [18,19].Then,ALP-OA/UA nanoparticles were prepared by an anti-solvent precipitation method to improve the aqueous solubility,stability and bioaccessibility of OA and UA.The structural features,physicochemical properties and stability of ALP-OA/UA nanoparticles were investigated by scanning electron microscopy (SEM),transmission electron microscopy (TEM),Fourier transform infrared (FTIR) spectroscopy,X-ray diffraction (XRD)analysis,derivative thermogravimetry (DTG) and dynamic light scattering.Finally,the combined anti-inflammatory effects of OA and UA along with ALPs in the form of ALP-OA/UA nanoparticles was evaluated using a CuSO4-induced zebrafish inflammation model,and the possible underlying mechanisms were explored by real time-polymerase chain reaction (RT-PCR).The success of ALPOA/UA nanoparticles in delivering an enhanced anti-inflammatory effects indicates the high potential of these nanoparticles in food and pharmacological applications.

2.Materials and methods

2.1 Materials

Dried slices ofA.lappa(Yanagawa-riso variety) were provided by Jiangsu Peixian Yiwei Co.,Ltd.(Jiangsu,China).Adult zebrafish were obtained from the Biology Institute of Shandong Academy of Sciences (Jinan,China).OA,UA and ibuprofen were purchased from Shanghai Yuanye Bio-Technology Co.,Ltd.(Shanghai,China).Dimethyl sulfoxide (DMSO) and CuSO4·5H2O were provided by Beijing Solarbio Science＆Technology Co.,Ltd.(Beijing,China)and Sinopharm Chemical Reagent Co.,Ltd.(Shanghai,China),respectively.Other chemicals,such as absolute ethanol,petroleum ether,ethyl acetate andn-butanol,were obtained from Tianjin Kaitong Chemical Reagent Co.,Ltd.(Tianjin,China).

2.2 Extraction and purification of ALPs

ALPs were extracted and purified following the procedure developed in our laboratory [15].Briefly,dried slices ofA.lappawere ground in a high-speed disintegrator,before two repeated aqueous extractions (at a water-to-tissue ratio of 10:1,mL/g) at 80 °C for 1.5 h.After the combined extracts were concentrated with a rotary evaporator at 60 °C,defatting and deproteinization were performed using 50% petroleum ether and Sevag reagent (chloroform:butyl alcohol=4:1,V/V),respectively.The resulting polysacchariderich liquid phase was subjected to precipitation using 4-fold volume of absolute ethanol,centrifugation and freeze drying using a SCIENTZ-12N freeze dryer (Ningbo SCIENTZ Biotechnology Co.,Ltd.,Ningbo,China).The lyophilized ALPs were used for subsequent experiments.

2.3 Preparation of ALP-OA/UA nanoparticles

ALP-OA/UA nanoparticles were produced according to the method of Li et al.with some modifications [20].Briefly,0.25 g of the ALP powder and a total of 10 mg of OA+UA (at different ratios)were added to 50 mL of deionized water and 10 mL of 70% ethanol,respectively,before both were subjected to a 30-min ultrasound treatment to facilitate complete dissolution.Afterwards,10 mL of the OA+UA solution was added dropwise to 50 mL of ALP solution under magnetic stirring at 300 r/min for 30 min (a weight ratio of ALPs to OA+UA solution=50:1).The resulting mixture was concentrated using a rotary evaporator operated at 42 °C,and then centrifuged using a TGL 16 centrifuge (Changsha Yingtai Instrument Co.,Ltd.,Changsha,China).The supernatant was collected and freeze dried (termed as ALP-OA/UA nanoparticles).

2.4 Characterization of ALPs and ALP-OA/UA nanoparticles

2.4.1 Microscopic analysis

Morphological features of the dried ALPs and ALP-OA/UA nanoparticles were examined by a SEM Zeiss-SUPRATM55 microscope (Oberkochen,Germany).The dried samples were deposited directly on the specimen stub and coated with gold using a sputter coater (EMS7620,New Jersey,USA) under vacuum.Images were acquired at an acceleration voltage of 5 kV and at magnifications of 5 000× and 50 000×.

Further TEM examinations were performed to reveal the fine structures of ALPs and ALP-OA/UA nanoparticles.ALP or ALP-OA/UA samples were mixed with appropriate amounts of deionized water to prepare well-dispersed suspensions.A portion of each suspension was then immediately withdrawn and dropped onto a copper grid (pore size: 250 mm) and left to dry under ambient conditions.Then,a drop of 2% phosphotungstic acid solution was added to allow staining for 10 min and drying naturally,before examinations using a FEI TalosTMF200 TEM (Thermo Fisher Scientific Inc.,USA) at an accelerating voltage of 80 kV.

2.4.2 FTIR-ATR spectroscopy analysis

Structural characterization of ALPs and ALP-OA/UA nanoparticles was performed using a Thermo Nicolet IS10 FTIR spectrometer equipped with a universal ATR accessory (Thermo Fisher Scientific Inc.,USA).Approximately 3 mg of dried sample was subjected to scanning (64 scans) in the wavenumber range of 4 000 to 400 cm-1at a resolution of 4 cm-1,with each test sample being analyzed in triplicate.The background spectrum of air was obtained for subtraction.ATR correction was performed using OMNIC 8.2.0.387 software.

2.4.3 XRD analysis

XRD patterns of ALPs and ALP-OA/UA nanoparticles were collected on an EMPYREAN X-ray diffractometer (Panalytical B.V.,Netherlands),equipped with a Cu Kα radiation source.Data were acquired over the diffraction angle (2θ) range of 5°–80° at 25 °C and a scan rate of 4°/min.The crystal structure of the polysaccharide samples was deduced from the XRD spectra.

2.4.4 Particle size and zeta potential

The particle size and zeta potential of ALPs and ALP-OA/UA nanoparticles were determined using an Zetasizer Nano ZS laser nanometer particle size analyzer (Malvern,Worcestershire,UK).The polysaccharide-containing sample was reconstituted in deionized water to yield a dispersion at the appropriate concentration before the measurements at 25 °C.

2.4.5 Thermal analyses

The thermodynamic properties of ALPs and ALP-OA/UA nanoparticles,including thermal gravity (TG),DTG and differential scanning calorimetry (DSC) examinations,were measured using a simultaneous thermal analyzer (Netzsch STA 449F3 Jupiter,Germany).The lyophilized sample powder (3–5 mg) was added into a standard aluminum pan and hermetically sealed,before being heated from 30 °C to 600 °C at an increasing rate of 10 °C/min under nitrogen gas at a flow rate of 20 mL/min.The empty aluminum pan was used as the reference.

2.4.6 Encapsulation efficiency (EE) and loading capacity (LC)

The EE and LC related to ALP-OA/UA nanoparticles were determined according to the method described by Li et al.[20].Briefly,an aliquot (2 mL) of nano-dispersions was mixed with 2 mL of methanol,and the mixture was centrifuged to obtain the supernatant.Then,the supernatant was filtered using a 0.22 μm millipore filter before the analysis by high-performance liquid chromatography (HPLC).The amounts of OA and UA were determined using a Shimadzu LC-20A HPLC system equipped with a diode array detector (Shimadzu,Kyoto,Japan) at a wavelength of 210 nm.A symmetry C18column (Waters,4.6 × 250 mm,5 μm;set at 30 °C) and a mobile phase consisting of 0.2% acetic acid solution and methanol (13:87,V/V;flow rate: 1.00 mL/min) were used.The injection volume was 10 μL.The EE and LC were evaluated according to the following equations.

2.5 Stability of ALP-OA/UA nanoparticles under different conditions

2.5.1 pH

The pH of the freshly prepared nanoparticle dispersions was adjusted to 2,3,4,5,6,7,8 and 9 with 0.1 mol/L HCl or 0.1 mol/L NaOH and stabilized for 24 h.Then,the appearance of the different nanoparticle systems was observed,and the particle size,zeta potential and polydispersity index (PDI) were measured using a Zetasizer Nano ZS laser nanometer particle size analyzer to evaluate their pH stability [21].

2.5.2 Temperature

As described by Song et al.,freshly prepared nanoparticles were dispensed into 10 mL-sealed vials and then incubated in a water bath at different temperatures (30,40,50,60,70,80 and 90 °C)for 1 h [21].After the samples were cooled to room temperature at (25 ± 2) °C,their particle size and zeta potential were determined to analyze the effect of temperature on the stability of ALP-OA/UA nanoparticles.

2.5.3 Ionic concentration

Aliquots of ALP-OA/UA nanoparticle dispersions were mixed with different concentrations of NaCl solutions to final concentrations of 0,25,50,75,100,150,200 and 250 mmol/L,respectively.After incubation overnight at 25 °C,the ionic stability of different nanoparticles was evaluated according to the changes in appearance,particle size,PDI and zeta potential [22].

2.5.4 Storage time

To obtain the storage stability of different nanoparticles,freshly prepared ALP-OA/UA nanoparticle dispersions were stored continuously at 25 °C for 30 days under light-proof conditions,with the addition of sodium azide (1:5,g/L) to prevent microbial spoilage.The particle size and zeta potential of the nanoparticles were recorded every 5 days.

2.6 Evaluation of the combined anti-inflammatory effects of ALPs,OA and UA in the form of ALP-OA/UA nanoparticles using a zebrafish model

2.6.1 Effect of different ratios of OA and UA on CuSO4-induced inflammation responses in the zebrafish model

Normal transgenic zebrafish (Tg: zlyz-EGFP) species were raised at 28 °C under a light/dark cycle of 14/10 h,withArtemia salinaused as the regular feed.After healthy and sexually mature individuals were placed in the breeding tank at a female-to-male ratio of 1:2 in the late night,the naturally developed embryos at 72 h post-fertilization(hpf) were selected under a stereomicroscope (AXIO Zoom.V16,Carl Zeiss,Germany) for subsequent experiments.

The obtained zebrafish embryos were transferred to individual wells of 24-well plates and randomly divided into eight groups (n=15/well): The blank control group (fresh culture water),the model group (fresh culture water),the positive control group (treated with 20 μmol/mL ibuprofen) and five OA+UA treatment groups (OA and UA at the total doses of 1,5 and 10 μg/mL in ratios of 100% OA,75% OA+25% UA,50% OA+50% UA,25% OA+75% UA and 100% UA,respectively).Then,all treatment groups were placed in an illuminated incubator (28 °C) for 3 h to allow embryo development.After the light-proof administration of 40 μmol/L CuSO4for 1 h to all groups (except for the control group),the inflammatory response was examined and recorded under a SZX16 fluorescence microscope(Olympus,Japan),and the number of macrophages was quantified using the Image-Pro Plus software.

2.6.2 Anti-inflammatory effects of ALPs and ALP-OA/UA nanoparticles on CuSO4-induced inflammation in zebrafish model

As described in Section 2.6.1,healthy zebrafish embryos at 72 hpf were selected under a stereomicroscope and transferred to individual wells of 6-well plates (n=30/well).All treatments provided were 5 mL of embryo medium containing ALPs and ALPOA/UA nanoparticles at final doses of 10,100 and 200 μg/mL,whilst ibuprofen was used for the positive control group,and the embryo culture medium was used for the model group.Each group was set up in triplicate.After incubation in an illuminated incubator at 28 °C for 3 h,all the zebrafish embryos were intervened by 40 μmol/L CuSO4solution for 1 h in the dark,except for the blank group (which contained only fresh culture water).Finally,the zebrafish larvae were monitored by the fluorescence imaging technique using a fluorescence microscope (Olympus,IX51,Tokyo,Japan),and the number of inflammatory cells was counted using the Image-Pro Plus software.

2.6.3 Total RNA extraction and RT-PCR

After OA+UA administration,zebrafish larvae were washed with fresh water,and their total RNA was extracted using a RC101FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme Biotechnology,Nanjing,China) according to the manufacturer’s instructions.The obtained RNA samples were subjected to reverse transcription for the synthesis of complementary DNA (cDNA) using a HiScript?III RT SuperMix for qPCR (Vazyme Biotechnology,Nanjing,China).Subsequently,the expression levels of inflammation-related genes (Table S1,Shanghai BioSune Biotechnology Co.,Ltd.) were evaluated by RT-PCR.

The reaction system consisted of 10 μL of 10 × ChamQ Universal SYBR qPCR Master Mix,0.4 μL of forward and reverse primers(10 μmol/L),1 μL of cDNA and 8.2 μL of ddH2O (a total of 20 μL).RT-PCR was performed in a Roche LightCycler 96 RT-PCR system under the following conditions: pre-denaturation at 95 °C for 30 s,40 cycles of 95 °C for 10 s and 60 °C for 30 s,followed by 1 cycle of 95 °C for 15 s,60 °C for 60 s and 95 °C for 15 s.Theβ-actin was used as the internal control,and the expression of target genes was determined by the comparative 2?ΔΔCtmethod.

2.7 Statistical analysis

Experimental data were reported as mean ± standard deviation(SD).The significance of differences was evaluated using one-way analysis of variance (ANOVA) followed by Tukey’s test (atP<0.05,0.01 or 0.001) using the SPSS software.

3.Results and discussion

3.1 Screening the optimal ratio between OA and UA in a CuSO4-induced zebrafish inflammation model

The number of inflammatory cells migrating to the lateral line in zebrafish larvae of the CuSO4-treated model group was increased significantly (P<0.001;by 17.6-fold) compared with the blank control group (Fig.1A).The administration of ibuprofen or a combination of OA and UA facilitated the return of macrophages,indicating their abilities to alleviate the inflammatory response.Ibuprofen led to a 42.05% decrease in the level of macrophages,and differences in the dose of the OA-UA combinations and the OA-to-UA ratios resulted in different but significant effects on the migration of macrophages (Fig.1B).The most effective treatment to alleviate the inflammation response was that with the dose of 1 μg/mL for 50% OA+50% UA,followed by that with the dose of 1 μg/mL for 25% OA+75% UA and that with the dose of 1 μg/mL for 75% OA+25% UA.Their treatments resulted in a decrease by 51.14%,44.32% and 42.61%,respectively,in fluorescence spots(representing macrophages) of zebrafish embryos,compared with the model group (P<0.001).In comparison,only a decrease by 27.27% –41.67% and 7.95% –38.07%,respectively,was observed for the sole use of OA or UA at doses of 1,5 or 10 μg/mL (P<0.05,0.01 or 0.001).These results indicated an enhanced anti-inflammatory effect between OA and UA.Therefore,the administration of an OA-UA combination (especially at the mass ratio of 1:1) was more effective than the administration of OA alone or UA alone in relieving the inflammation damage caused by CuSO4.Accordingly,the OA-to-UA mass ratio of 1:1 was used for subsequent nanoparticle preparation.

Fig.1 Effect of different ratios of OA and UA on CuSO4-induced inflammatory response in zebrafish: (A) Representative images of zebrafish treated with CuSO4 and different OA-to-UA ratios;(B) Migration of macrophages to the lateral line revealed by Image-Pro Plus software.Data are represented as the mean ± standard deviation.***P <0.001,**P <0.01 and *P <0.05 vs.the model group,###P <0.001 vs.the control group.

Accumulating evidence has confirmed the anti-inflammatory activity of UA and OA through multiple pathways,including common or exclusive mechanisms.UA was found to attenuate CCl4-induced hepatic dysfunction and histopathologic changes,alleviate CCl4-induced oxidative damage by inhibiting the production of reactive oxygen species in the liver and suppress inflammatory response through MAPK (lowering the activation of JNK,p38 MAPK and extracellular regulated protein kinases (ERK)) and NF-κB pathways(reducing the translocation of NF-κB p65 from the cytosol to the nuclear fraction,down-regulating the protein expression of TNF-α,IL-1β and COX-2) [23].In another study,UA administration significantly ameliorated acute inflammation by reducing renal injury and xylene-induced ear oedema and protected against LPS-induced acute kidney injury in BALB/C mice.This might be due to the reduced secretion of inflammatory factors TNF-α,IL-6 and IL-1β in macrophages,inhibition of TLR4/MyD88 pathway and up-regulation of macrophage autophagy by increasing the expression of both LC3B and Beclin-1 [24].In terms of OA,it significantly attenuated the proinflammatory response in mice with spinal cord injury via inhibiting the activation of MAPKs and NF-κB signaling pathways,mainly through the down-regulation of p38,JNK,IKKα,IκBα and NF-κB [5].Similar anti-inflammatory activity appeared in mice with DSSinduced colitis: abnormal colon shortening,macroscopic scores and myeloperoxidase activity were effectively alleviated via inhibiting Th17 cell differentiation,down-regulating the activation of NF-κB and MAPKs and the expression of TNF-α,IL-1β and IL-17,and improving Treg cell differentiation and IL-10 expression [6].

It was reported that the combinations of drugs or bioactive components with different mechanisms might be more conducive to synergistic effects [25].We hypothesized that UA and OA shared both common and exclusive anti-inflammatory pathways when used together,leading to enhanced health benefits.Our research team identified 23 key common targets with anti-inflammatory pathways between UA and OA based on Swiss Target Prediction,TCMSP,PubMed,CTD,Drug Bank and GeneCards.The major genes involved were CASP8,TNFRSF1A and TLR9,which further supported our hypothesis.However,future research was needed to explore the specific mechanisms underlying the synergistic effects of antiinflammatory activity.

3.2 Characterization of ALP-OA/UA nanoparticles

3.2.1 Morphological features of ALPs and ALP-OA/UA nanoparticles

As shown in Fig.S1A and S1B,ALPs appeared as irregular and extended discoids,while ALP-OA/UA nanoparticles looked like a mass full of very small,connected and irregular pieces under 5 000×magnification.The surface ofδ-orγ-inulin was previously described as discoid-like morphology [26].Under a higher magnification(50 000×;Fig.S1C and S1D),ALPs appeared as aggregates of small particles,while ALP-OA/UA nanoparticles contained a mixture of sheet-like structures and small particles in chain-like networks.TEM images (Fig.S1E and S1F) showed the same morphologies as seen in the high magnification SEM images: ALPs looked like an aggregated mass,while ALP-OA/UA nanoparticles appeared as a pore-containing network made up of interconnected spherical beads.The morphological differences between ALPs and ALPOA/UA nanoparticles were anticipated to influence their physicochemical and biological properties.As reviewed by Kinnear et al.,the morphological differences might potentially affect nanobiological interfaces and thus dictate their interactions with single cells and eventually whole organisms [27].In vitro,the geometric characteristics of nanoparticles were tightly related to particle-cell interactions.In particular,the morphological features influenced the radius of curvature of the particles in contact with the cell surface,which was critical in determining the amount and rate of particle phagocytosis.Within a certain scope,rod-shaped particles seemed to be more easily absorbed compared with spherical particles due to more contact with the cell surface [27].In vivo,oral administration required that nanoparticles continued to pass through the gastrointestinal tract and cross the mucosal barrier.Compared with spherical nanoparticles,there was a higher intestinal surface coverage and greater penetration and accumulation in the small intestine for rod-shaped nanoparticles,which resulted from the porosity of mucus and the shape-mediated diffusion/movement of anisotropic nanorods in the gel-like network [28].Therefore,ALP-OA/UA nanoparticles with higher aspect ratios possessed more contact with the cell surface and more absorption in the intestinal tract.Furthermore,the porecontaining network structure of ALP-OA/UA nanoparticles also facilitated human gastrointestinal digestion by increasing the contact area of nanoparticles [29].

3.2.2 FTIR analysis

The FTIR spectra in Fig.2A showed typical IR bands of polysaccharides or pentacyclic triterpenoids: 3 230–3 280 cm-1(O-H stretching vibrations of hydroxyl groups),2 930 cm-1(C-H stretching vibrations,including CH,CH2and CH3groups),1 740 cm-1(C=O stretching vibration of esterified carboxyl groups,-COOR),1 420 cm?1(C-H deformation of CH2/CH3groups of aromatic and aliphatic structures,or O-H bending),1 200–800 cm?1(C-O,O-C-O and C-O-C stretching/bending of the glycosidic linkages or rings in polysaccharides) [15,30].The spectra of ALPs and ALP-OA/UA nanoparticles showed the same characteristic signals.The loaded OA and UA were not seen in the spectrum of ALP-OA/UA nanoparticles due to their low concentration and overlap with the characteristic signals of ALPs.A slight shift from 3 270 cm-1for ALPs to 3 330 cm-1for ALP-OA/UA nanoparticles,along with a slight decrease in the intensity of this signal for ALP-OA/UA (compared with ALPs) indicated the involvement of O-H of hydroxyl groups(likely hydrogen bonding).A significant increase in the signal intensity at 1 030 cm-1for ALP-OA/UA nanoparticles compared with ALPs suggested that the loading of OA and UA onto ALPs had affected the C-O/O-C-O stretching vibration (likely in pyranose ring form) [31].As shown in Fig.S2,the FTIR spectra of the physical mixture of ALPs,OA and UA were very similar to those of ALPs,but were significantly different from those of ALP-OA/UA nanoparticles:there was no band shift and intensity drop around 3 300 cm-1and no signal intensity at 1 030 cm-1.Therefore,the encapsulation of OA and UA by ALPs involved the O-H of hydroxyl groups (likely hydrogen bonding) and the C-O groups or O-C-O structure.

Fig.2 (A) FTIR spectra of OA,UA,ALPs and ALP-OA/UA nanoparticles.(B) XRD patterns of OA,UA,ALPs and ALP-OA/UA nanoparticles.(C) Particle size distribution of ALPs and ALP-OA/UA nanoparticles.(D) HPLC profiles: (a) Burdock polysaccharide (ALP) aqueous solution;(b) OA UA aqueous solution(mass ratio 1:1);(c) OA-UA methanol solution (mass ratio 1:1);(d) ALP-OA/UA nanoparticle aqueous solution.

3.2.3 XRD analysis

As presented in Fig.2B,the non-encapsulated OA and UA occurred as crystals.Three significant diffraction peaks at 2θof 21.67°,17.73° and 12.00° in the XRD pattern of ALPs indicated the semi-crystalline nature.Such characteristic peaks were previously found in the XRD pattern of natural inulin [16].After the loading of OA and UA onto ALPs,the intensities of these three diffraction peaks of ALPs were significantly reduced,and the typical diffraction peaks of OA and UA did not appear in the XRD pattern of ALP-OA/UA nanoparticles (the nature of OA and UA changed from crystalline to a more amorphous structure).Therefore,ALP-OA/UA nanoparticles were more amorphous than ALPs.The encapsulation of OA and UA by ALPs decreased greatly their crystallinity likely via non-covalent interactions between the widely distributed functional groups of ALPs and the two triterpenic acids,and physical entrapment/compartment of OA and UA molecules inside the pores of ALP network [32].Noncovalent interactions,mainly hydrogen bonding,are the key force to form crystals.FTIR spectra revealed that the preparation of ALP-OA/UA nanoparticles involved hydrogen bonding interaction between the widely distributed functional groups of ALPs and the two triterpenic acids.This blocked the rotational movement of hydrogen bonds and their interactions,thus affecting the formation of crystals [33].Furthermore,the formation of crystals required an ordered arrangement of particles.According to the typical conformation proposed by Vereyken et al.,there were two favorable conformations of the inulin-type fructans with DP of 10: one having right-handed helical structure with 4,5,or 6 units per turn and an initial dihedral angle (ω) at 60°,and the other one with a zigzag conformation andωat 180° for all Fru-(2→1)-Fru linkages [34].They were relatively flexible,especially at both ends.After the encapsulation of OA+UA,two triterpenic acids were embedded in the helical structure of inulintype fructans.Therefore,the physical entrapment/compartment of OA and UA within the pores of ALP network prevented free rotation and distortion of ALPs to form orderly arranged crystals.The destruction of crystalline structures,along with the increase in aqueous solubility induced by nanonization,would likely improve the bioavailability of OA and UA.

3.2.4 Particle size and zeta potential

As shown in Fig.2C,both ALPs and ALP-OA/UA nanoparticles had a narrow and unimodal distribution with their average diameters around 178.2 nm and 199.1 nm,respectively.The incorporation of OA and UA into ALPs increased slightly the average particle diameter,which might be attributed to the crystalline nature of OA and UA and their relatively high molecular weight (456.7 g/mol),implying the interaction between ALPs and triterpenic acids [35].Li et al.[20]found a significant increase in the size of zein nanoparticles and zein/soluble soybean polysaccharide composite nanoparticles after they encapsulated triterpene acids.The diameters of ALPs and ALP-OA/UA nanoparticles determined by the particle size analyzer were larger than the diameter results obtained by TEM.Such a difference might result from the differences in the preparation process and nature of the tested sample (dried samples for TEM analysis and aqueous dispersions for measurements by the particle size analyzer).

The zeta potential of nano-dispersions is the potential difference between the continuous phase and the fluid-stabilized layer attached to the dispersed particles rather than the charge carried by the molecules or generated by ionization,representing the electrostatic behavior of colloidal particles in a medium [36].It was reported that inulin as a non-ionic polysaccharide with abundant hydroxyl groups exhibited a negative zeta-potential (-5.78--10 mV) in an aqueous solution [37,38].In this study,ALPs had a zeta-potential of (-9.65 ± 0.88) mV.In comparison,ALP-OA/UA nanoparticles had a significantly increased zeta-potential of (-7.15 ± 1.17) mV,likely due to the encapsulation of OA and UA with lower negative charges (P<0.05;zeta-potential: (-3.86 ± 0.11) and(-5.92 ± 0.13) mV,respectively).It was presumed that the loading of OA and UA onto ALPs altered the forces (such as van der Waals forces and hydrophobic interactions) and charge distribution on the polysaccharide surface and the resulting dispersed nanoparticles,thereby affecting the level of zeta potential.Therefore,ALPs and OA+UA with different charges could be co-assembled into nanoparticles during the encapsulation process [39,40].Inulintype fructans have gained great interest due to their ability to form nanoparticles and potential application as nano-carriers for drug delivery.The enzymatically synthesized high molecular weight(HMW) inulin was reported to self-assemble to form stable spherical nanoparticles,with an average diameter of (112 ± 5) nm [37].Furthermore,Zhang et al.reported the preparation of inulin-ibuprofen nanoparticles prior to the synthesis of RGD peptide (arginineglycine-aspartic acid) conjugated inulin-ibuprofen nanoparticles for targeted delivery of epirubicin [41].The above evidence confirmed the possibility of nanoparticles composed of inulin with negatively charged small molecule components or inulin itself.

3.2.5 Thermal properties

Thermal analysis of nanoparticles provides helpful information about the thermodynamical properties and end-use performance of materials.Furthermore,some structural features could be reflected by thermodynamic behavior,such as molecular weight distribution range and molecular arrangement.In general,the thermodynamic characteristics of ALPs and ALP-OA/UA nanoparticles resembled,although small differences were still found in some temperature regions (Fig.3).In terms of TG analysis,the first thermal decomposition stage (where the loss of volatiles and retained moisture took place) occurred in the range of 30.00-214.67 °C and 30.00-207.33 °C for ALPs and ALP-OA/UA nanoparticles,respectively,with ALP-OA/UA nanoparticles having a lower rate of mass loss than ALPs.ALPs and ALP-OA/UA nanoparticles essentially exhibited the same behaviors in the second stage (215-305 °C,where about 45% mass loss took place owing to decarboxylation and thermal decomposition) and third stage (>305 °C,where formation of char and further thermal degradation of char were possible).In DTG curves,major differences between ALPs and ALP-OA/UA nanoparticles were found in the regions of 40-130 °C and 220-280 °C),with both having a maximum difference in water loss occurring around 82 °C (the first endothermic peak).The maximum decomposition rates occurred at 237.17 °C and 275.50 °C for ALPs,and 228.00 °C and 279.67 °C for ALP-OA/UA nanoparticles.Silva et al.[42]found that inulin exhibited similar thermodynamic patterns: the thermal decomposition process of the inulin with a degree of polymerization (DP) ≥10 and DP ≥23 underwent three stages,with the greatest weight loss occurring at approximately 240–270 °C.It should be noted that the weight loss of ALP-OA/UA nanoparticles was less than 10% when the temperature reached 207.33 °C,indicating their potential application in high-temperature processed foods (such as ultra-high temperature sterilization).In terms of DSC curves,the main differences between ALPs and ALPOA/UA nanoparticles were mainly found at both ends (<220 °C or>310 °C).ALP-OA/UA nanoparticles had only one valley at 230 °C(decomposition temperature),whilst ALPs had a main valley at 230 °C (decomposition temperature) and a very small valley around 190 °C (likely isomerization temperature).Pure OA and UA were reported to have a sharp endothermic peak (the characteristic meeting point of anhydrous crystalline) at 312 °C and 289 °C,which might contribute to the large differences between ALPs and ALP-OA/UA nanoparticles in the region >310 °C [43].According to the thermodynamic data of ALPs and ALP-OA/UA nanoparticles,the loading of OA and UA onto ALPs did not change the main structural features of the polysaccharides,but the slight differences implied the successful encapsulation of OA and UA by ALPs.The above results revealed that ALPs and ALP-OA/UA nanoparticles had similar thermal properties,with ALPs likely experiencing a bit more mass loss at temperatures <220 °C.

Fig.3 Thermodynamic analyses of burdock polysaccharides (ALPs) and ALP-OA/UA nanoparticles.(A) TG curves;(B) DTG curves;(C) DSC curves.

3.3 EE and LC of ALP-OA/UA nanoparticles

As shown in Fig.2D,there were two peaks (retention time:21–23 min) in the HPLC chromatograms of the OA-UA methanol solution and aqueous solution of ALP-OA/UA nanoparticles,which corresponded to OA and UA,respectively,whilst these two peaks were absent in the HPLC chromatogram of the OA-UA aqueous solution.Methanol was more effective than water in solubilizing OA and UA,due to the polarity matching between methanol and those of oleanolic acid and ursolic acid [44].In agreement with the previous finding that nanonization could increase solubility,OA and UA in the form of ALP-OA/UA nanoparticles became more extractable(a total of (9.604 ± 0.74) mg/g of OA and UA was detected) [7].Nanoparticles have large specific surface areas therefore can promote the dissolution and diffusion of substances [40].According to the contents of OA and UA in different systems,the EE and LC of the ALP-OA/UA nanoparticles were (48.98 ± 3.77)% and (0.96 ± 0.07)%,respectively.

3.4 Stability of ALP-OA/UA nanoparticles

3.4.1 pH

Nanoparticles will undergo drastic pH changes in the gastrointestinal tract as delivery carriers of functional components after being ingested by the human body.Accordingly,it is important to assess the pH stability of composite nanoparticles for their practical application.As described in Fig.4A,the particle size of ALP-OA/UA nanoparticles showed a decreasing trend with the increase of pH from 2.0 to 9.0,and all values were less than 300 nm.The largest particle size (293.35 nm) appeared at pH 2,which was significant reduced to 186.63–208.40 nm at pH 6–9 (P<0.05).This indicated that ALP-OA/UA nanoparticles displayed better anti-aggregation stability in the higher pH range.The trend of zeta potential was similar to particle size.It could be observed that the negative zeta potential of ALP-OA/UA nanoparticles were significantly increased (with pH from 2 to 9) until it reached the highest value of ?13.77 mV at pH 9(P<0.05).This might be due to the fact that deprotonation phenomenon became gradually apparent when the pH range was higher than the pKa value of -O? in ALPs,which led to smaller particle size under enhanced surface charge and repulsion.Furthermore,the change in narrow particle size distribution of ALP-OA/UA nanoparticles at different pH also supported the experimental results of particle size.At pH 2.0,the PDI of ALP-OA/UA nanoparticles ranged 0.35–0.40,which was dramatically reduced to 0.23 at pH 6 (P<0.05),and there was a slight decrease with further increase in pH.Notably,the PDI of all ALP-OA/UA nanoparticles was less than 0.25 after pretreatment at pH 6.0–9.0,with uniformly stable and clear appearance.At pH 9.0,ALP-OA/UA nanoparticles had the smallest particle size (186.6 ±1.8) nm and PDI (0.18 ± 0.02),as well as the largest negative zeta potential (?13.77 ± 0.60) mV.In general,the smaller PDI values reflect the more stable nanoparticle dispersion system,where nanoparticles with PDI below 0.3 are considered homogeneous and those in the range of 0.3–0.4 are considered acceptable [33,45].Taken together,the composite nanoparticles exhibited excellent stability in the pH range of 6?9,and the smaller particle size and PDI suggested that alkaline conditions promoted the stabilization of ALP-OA/UA nanoparticles due to the driver of larger negative zeta potential.

3.4.2 Temperature

As thermal treatment is widely used in food processing,the excellent heat resistance of nanoparticles can protect the delivered bioactive components from being destroyed [21].Fig.4B showed the particle size,PDI and zeta potential of ALP-OA/UA nanoparticles after pretreatment at 30-90 °C.It was clear that the particle size of the nanoparticles pretreated at 30?90 °C did not change greatly and fluctuated around 236 nm.There was a relatively high particle size at 50 °C (258.1 nm) and 80 °C (241.3 nm),and a relatively small particle size at 70 °C (215.4 nm) for ALP-OA/UA nanoparticles.A remarkably similar change pattern occurred in the PDI-temperature profile.At different pretreatment temperatures,the PDI ranged from 0.24-0.38,and nanoparticles with larger particle sizes tended to have higher PDI.Compared with the ALP-OA/UA nanoparticles pretreated at 30 °C,the thermal treatment at 40?90 °C resulted in a slightly wider particle size distribution,but within an acceptable range.Furthermore,the zeta potential of the nanoparticles was insensitive to the change in temperature apart from 80 °C,with essentially the same value at all treatment temperatures (around 11 mV),indicating that the zeta potential (electrostatic repulsive force) was not responsible for the change in particle size of the nanoparticles.As the treatment temperature rose,the violent movement of nanoparticles might increase the chance and strength of collisions,thus exacerbating the aggregation.Meanwhile,ALP was an inulin-type fructan with good thermal stability,and the stretching of its molecular chain might be enhanced at higher temperatures.These two effects led to fluctuating variations in the particle size and PDI of ALP-OA/UA nanoparticles.As a result,limited and acceptable fluctuations in the particle size and PDI of ALP-OA/UA nanoparticles (no significant changes in appearance) were observed after pretreatments at different temperatures,showing certain thermal stability.

Fig.4 Stability of nanoparticles at different (A) pH,(B) temperature,(C) ion concentration conditions and (D) storage time at room temperature and 4 °C.

3.4.3 Ionic concentration

Composite nanoparticles experience various complex ionic environments during food processing and digestion and absorption in the human gastrointestinal tract.As shown in Fig.4C,ALP-OA/UA nanoparticles were highly susceptible to salt ions,especially at high NaCl concentrations.Although the particle size of the composite nanoparticles increased from 208.6 nm to 2 140.6 nm as the NaCl concentration reached 100 mmol/L,there was no significant change in the appearance compared with the freshly prepared nanoparticles.At the NaCl concentration of 150 mmol/L,the particle size of ALP-OA/UA nanoparticles steeply increased to over 3 000 nm(P<0.05),which resulted in aggregation and precipitation at the bottom of the tubes.The PDI was positively correlated with the particle size of nanoparticles: a sharp rise followed by a slow increase (>0.9 when the salt concentration was greater than 100 mmol/L).This suggested that the narrow particle size distribution of freshly prepared nanoparticles was significantly increased due to the enhanced ionic strength.The zeta potential measurement could partially explain this phenomenon [21].As the salt ion concentration increased,the negative zeta potential of ALP-OA/UA nanoparticles continued to decrease.The enhanced adsorption of Na+on the composite nanoparticles produced electrostatic shielding or neutralized the surface charge of the particles,which weakened the electrostatic repulsion between the nanoparticles and thus resulted in aggregation [21,46].Notably,these results also revealed that the electrostatic repulsion dominated in the interaction that stabilized nanoparticles.Nanoparticles could resist aggregation due to steric hindrance (caused by electrostatic repulsion)overcoming hydrophobic and Van der Waals interactions between particles until the particle size exceeded a critical value at enhanced NaCl concentrations [22].

3.4.4 Storage time

The storage stability of the composite nanoparticles is closely related to the potential for commercial application,and the changes in particle size,PDI and zeta potential at room temperature and 4 °C were presented in Fig.4D.A slight decrease in the particle size of nanoparticles (from the initial 199.1 nm to 190.9 nm on day 30) was observed during storage at room temperature for 30 days,accompanied by a mild increase in PDI (from the initial 0.20 to 0.23 on day 30).This suggested that the nanoparticle dispersions had a good stability during storage of 30 days.In contrast,an increasing trend was observed in both particle size and PDI of the nanoparticles at 4 °C,and they were generally higher than those at room temperature.Interestingly,the particle size of nanoparticles was less than 220 nm,the PDI detected at each time point remained around 0.24,and the nanoparticle dispersions were all clear and transparent with no obvious precipitation under two storage conditions.This again proved that the temperature only produced little effect on the nanoparticles,which was in agreement with the results of the thermal stability of nanoparticles.The zeta potential is related to the aggregation tendency of the particles in solution and thus reflects the stability of the nanoparticle system.The negative zeta potential of ALP-OA/UA nanoparticles showed a rising trend over 20 days of storage at room temperature and 4 °C,indicating a continuous increase in electrostatic repulsion.After 25 days of storage,a sharp decrease and then a steep increase in the negative zeta potential of the nanoparticles was observed.Nevertheless,the appearance,particle size and PDI remained stable at this time.This might be due to the presence of other interactions between the nanoparticles.Consequently,ALP-OA/UA nanoparticles had an excellent stability at room temperature and 4 °C for a shelf life of 30 days.

3.5 Evaluation of the anti-inflammatory activities of ALPs and ALP-OA/UA nanoparticles and the underlying mechanisms

3.5.1 Anti-inflammatory effects in a CuSO4-induced zebrafish inflammation model

Fig.5 showed that ALPs and ALP-OA/UA nanoparticles at different doses (10,100 or 200 μg/mL) exhibited significant antiinflammatory activities in a CuSO4-induced zebrafish inflammation model.The administration of CuSO4caused a large rise (from 2.60 to 29.14 compared with the blank control) in the number of inflammatory cells migrating to the lateral line of zebrafish larvae(P<0.001),indicating the successful establishment of a CuSO4-induced zebrafish inflammation model.After the intervention with ibuprofen,ALPs and ALP-OA/UA nanoparticles,CuSO4-induced migration and subsequent accumulation of macrophages decreased greatly,indicating the alleviation of inflammation (i.e.the number of migrant inflammatory cells decreased by 60.20% in ibuprofentreated group compared with the model group;P<0.001).Both ALPs and ALP-OA/UA nanoparticles at doses of 10,100 and 200 μg/mL exhibited dose-dependent inflammation-suppressing effects,although these effects were smaller than that of ibuprofen.At the same dose,ALP-OA/UA nanoparticles had either a significantly higher (dose: 10 or 100 μg/mL) or insignificantly different (dose:200 μg/mL) anti-inflammatory activity,compared with ALPs (P<0.05).Therefore,the encapsulation of OA+UA with ALPs could allow the joint action between ALPs and OA+UA to alleviate the inflammation caused by CuSO4in zebrafish,with the greatest enhancement occurring at 100 μg/mL.

Fig.5 Alleviation of CuSO4-induced inflammatory response in zebrafish by burdock polysaccharides (ALPs) and ALP-OA/UA nanoparticles.(A) Representative images of zebrafish treated with CuSO4 and different ALPs or ALP-OA/UA treatments;(B) Migration of macrophages to the lateral line revealed by Image-Pro Plus software.Data are represented as the mean ± standard deviation.***P <0.001 vs.the model group,###P <0.001 vs.the blank control group.

Notably,there was a significantly higher alleviating performance on CuSO4-induced inflammatory damage after the administration of ALP-OA/UA nanoparticles (up to 54.90% reduction),compared with OA alone (up to 41.67% reduction),UA alone (up to 38.07% reduction) or OA-UA combination (at the mass ratio of 1:1,with a maximum 51.14% reduction) (P<0.05).Furthermore,another key advantage of ALP-OA/UA nanoparticles was the improved aqueous solubility of bioactive compounds (OA and UA).Although OA and UA dissolved in dimethyl sulfoxide had excellent protective effect against CuSO4-induced inflammation damage (Fig.1),little antiinflammatory effect was found in their aqueous solutions (due to poor aqueous solubility).In comparison,ALP-OA/UA nanoparticles exhibited desirable water solubility,and their aqueous solutions exerted significant anti-inflammatory activity.Therefore,ALPOA/UA nanoparticles not only significantly enhanced the antiinflammatory activity of OA and UA,but also improved their poor aqueous solubility.

3.5.2 Effect of ALPs and ALP-OA/UA nanoparticles on the gene expression of inflammation-related transcription factors and cytokines in zebrafish

To preliminarily explore the mechanisms underlying the antiinflammatory effects of ALPs and ALP-OA/UA nanoparticles,the expression levels of the transcription factors and cytokines of the NF-κB pathway in zebrafish were examined by RT-PCR [47,48].As presented in Fig.6,the expression levels of NF-κB2 (a zebrafish homolog of NF-κB),TNF-α,IL-1β and IL-8 of the model group increased by 1-to 4-fold compared with the blank control group(P<0.001).After the treatments with ALPs and ALP-OA/UA nanoparticles at doses of 10,100 and 200 μg/mL,the mRNA expressions of the four transcription factors and cytokines in zebrafish were significant down-regulated.Such changes were also found in mice suffering with colitis and in LPS-stimulated RAW264.7 macrophages [4,49].At the same dose,the mRNA expression levels ofNF-κB2andTNF-αin the ALP-OA/UA nanoparticle-treated group were lower than those in the ALP-treated group.The mRNA expression levels ofIL-1βandIL-8showed a different trend (ALPs ALP-OA/UA) for IL-1β or an equal effect (ALPs=ALP-OA/UA)for IL-8 at a dose of 200 μg/mL.The differences in the underlying mechanisms of anti-inflammatory activity of ALPs and ALP-OA/UA nanoparticles might result in different trends in the mRNA expression levels of these genes.After escaping hydrolysis in the upper gastrointestinal tract and entering the colon,ALPs were rapidly fermented by gut bacteria.The produced short chain fatty acids and other metabolites exerted anti-inflammatory activity through multiple pathways [17].In comparison,OA and UA could be absorbed directly into the bloodstream in the intestine and then transported throughout the body to exert biological activity [2].Furthermore,although ALPs and ALP-OA/UA nanoparticles were effective in restoring immune homeostasis (balance of pro-inflammatory cytokines (IL-1β,IL-6 and TNF-α) and anti-inflammatory cytokines (IL-10)),the difference in their primary targets might be responsible for the different regulation of the expression of the four genes.Moreover,the achievement of anti-inflammatory activity was time-dependent,and the different regulation of the mRNA expression of these genes might be attributed to time-dependent effects [48].

Fig.6 Effect of burdock polysaccharides (ALPs) and ALP-OA/UA nanoparticles on the gene expression levels of inflammatory-related transcription factors and cytokines in zebrafish.(A) NF-κB2;(B) TNF-α;(C) IL-1β;(D) IL-8.Data are represented as the mean ± standard deviation.***P <0.001,**P <0.01 and*P <0.05 vs.the model group,###P <0.001 vs.the blank control group.

It was also observed that the alleviation of mRNA expression of these inflammation-related transcription factors and cytokines was not always dose-dependent,which was similar to other studies [48,50,51].In general,the bioactivity of natural products presented a parabolic pattern in response to dose,and there was an optimal working concentration.As the sample concentration increased from 10 μg/mL to 200 μg/mL,the higher inhibition of the transcription factors and cytokines indicated that the optimal working concentration was closer to 200 μg/mL (the apex of the parabola),while the trend of increasing and then decreasing effects for inflammation alleviation suggested that the optimal working concentration was closer to 100 μg/mL.Therefore,ALPs and ALP-OA/UA nanoparticles might exert antiinflammatory effects via the NF-κB signaling pathway.

4.Conclusion

The present study demonstrated the suitability of ALPs as natural wall materials for producing ALP-based nanoparticles to deliver OA and UA with poor aqueous solubility.The encapsulation of OA+UA with ALPs (an ALP:OA+UA weight ratio,50:1;optimal OA-to-UA ratio,1:1) decreased greatly their crystallinity,and allowed the joint action between ALPs and OA+UA to alleviate the inflammation caused by CuSO4in zebrafish via down-regulating the NF-κB signalling pathway and the gene expression of associated transcription factors and cytokines (TNF-α,IL-1β and IL-8).The greatest enhancement in the anti-inflammatory activity took place for ALPOA/UA nanoparticles dosed at 100 μg/mL.ALPs and ALP-OA/UA nanoparticles had similar functional groups and chemical bonds,particle size distributions (narrow and unimodal distribution),and thermal properties.The encapsulation of OA and UA by ALPs likely involved hydroxyl groups (for hydrogen bonding),C–O groups and O–C–O structures.Therefore,in addition to the anti-inflammatory activities brought by OA,UA and ALPs,the morphology,structure and physico-chemical properties (pH,temperature,salt solution and storage time) of the ALP-OA/UA nanoparticles enhanced the anti-inflammatory effect of CuSO4-treated zebrafish.Both ALPs and ALP-OA/UA nanoparticles are potential anti-inflammatory agents.In addition,ALP-based nanoparticles can serve as natural delivery systems for hydrophobic bioactive molecules in food and pharmaceutical preparations.

Conflict of interest

Declarations of interest: none.

Acknowledgements

This work was supported by the Shandong Provincial Natural Science Foundation of China (ZR2019BC100),Science,Education and Industry Integration Innovation Pilot Project of Qilu University of Technology (Shandong Academy of Sciences) (2020KJC-ZD10) and Incubation Program of Youth Innovation in Shandong Province.

Appendix A.Supplementary data

Supplementary data associated with this article can be found,in the online version,at http://doi.org/10.1016/j.fshw.2022.07.047.