The Influences of Antioxidants on the Stability of Coix Seed Oil

Liposomes Under Ultraviolet Irradiation

Yin Wang, Songbo Ma, Meilan Yuan

, Li Zhao and Chunqing Bai

College of Life Science, Jiangxi Science and Technology Normal University, Nanchang, China

* Correspondence Author: 1020140969@jxstnu.edu.cn

Keywords:

Liposomes, Coix Seed Oil, Antioxidant.

Abstract: In this research, coix seed oil (CSO) liposomes containing antioxidants were prepared by ethanol injection

method. The influence of types and concentrations of antioxidants on the physicochemical stability of

CSO liposomes irradiated by ultraviolet (UV) light was investigated in terms of particle size distribution

and malondialdehyde (MDA) production. The results showed that lipid peroxidation and liposomal

particle size change were induced by UV irradiation. The tert-butylhydroquinone (TBHQ) and

dibutyl-hydroxytoluene (BHT) exhibited better resistance on size change and peroxidation induced.

However, β- carotene exerted good anti oxidative activity at low concentration; the antioxidant effect was

weaken and even promoted oxidation at higher concentration. Although the antioxidant effect of

α-tocopherol was enhanced as the concentration increased, its influence on liposomal size varied and

dependent largely on exposed time. In addition, the lower MDA value of CSO liposomes than that of

control indicates the oil could supply anti oxidative activity against the peroxidation of liposomal

membrane. This research would supply good foundation for prolonging the shelf of CSO liposomes.

1 INTRODUCTION

Coix seed is a traditional herbal planted in many

Asian countries, such as Indian, China, and so on,

where the seed could be consumed as medicine and

food (Bai, 2019). According to literatures, the oily

abstracts named as coix seed oil was the main active

ingredients in the seed. Numerous experiments have

proved that the oil exhibited excellent antitumor,

anti-inflammation and analgesia activities (Zhu,

2017). However, the drawbacks of water

water-insolubility, low accessibility, combined with

poor oxidative stability significantly confined the

wide utilization of CSO. Thus, strategies that could

solve the above problems are needed to be thought

out.

To deal with these drawbacks of CSO, various

delivery systems (microcapsule, microsphere,

microemulsion, and liposomes) have been

developed by many researchers (

Nakhaei, 2021;

Chen, 2022)

. During the past several years, we also

carried out relative researches, and liposomes were

used to encapsulate CSO. The delivery system was

proved to be an efficient carrier that could supply

good protection for CSO from adverse environments

and promote controlled release of CSO in

gastrointestinal tract (Bai, 2019). However,

phospholipids, as the main membrane materials for

liposomes, contain certain amount of unsaturated

fatty acids. That means the bilayer is sensitive to

external environment (oxygen, lights, and so on) and

easily to be oxidized and decomposed, producing

harmful chemicals, including lysophospholipids etc.

Introducing antioxidant into liposomes was a useful

way to delay the degradation (

Rafaela, 2021;

Walker, 2017; Palmina, 2021)

. Usually, the

efficiency of the antioxidant activity was largely

dependent on the propertied of liposomes

themselves as well as the types and concentrations

of antioxidant used. However, nothing was known

about how to effectively protect liposomes

containing CSO.

In this sense, four common used antioxidants

(TBHQ, BHT, β-carotene, and α-tocopherol) were

chosen and encapsulated together with CSO to delay

the oxidant of CSO liposomes. The physical and

chemical stability of the liposomes during UV-light

exposure was monitored and determined in details.

This research would supply certain foundation for

the development of stable CSO liposomes.

Wang, Y., Ma, S., Yuan, M., Zhao, L. and Bai, C.

The Inﬂuences of Antioxidants on the Stability of Coix Seed Oil Liposomes Under Ultraviolet Irradiation.

DOI: 10.5220/0012001400003625

In Proceedings of the 1st International Conference on Food Science and Biotechnology (FSB 2022), pages 52-58

ISBN: 978-989-758-638-5

 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

2 MATERIALS AND METHODS

2.1 Materials

Egg yolk phospholipids and cholesterol were bought

from Shanghai Lanji Technology Development Co.,

Ltd (Shanghai, China). Coix seed oil was purchased

from Hecheng Sanxian Biotechnologies Co., Ltd.

(Guangzhou, China). TBHQ, BHT, α-tocopherol,

β-carotene, and 2, 2 -azobis(2-methypropionamidin)

dihydrochloride (AAPH) were kindly supplied by

Sigma-Aldrich (Shanghai, China). All other

chemicals used were of analytical grade.

2.2 Preparation of Liposomes

Liposomes were prepared by ethanol injection

method as described previously by us with slight

modification (

Bai, 2019)

. Briefly, weighted amounts

of antioxidants were dissolved in CSO, and diluted

with the oil to obtain a serial concentration of

antioxidants/CSO solution. The mixture, egg yolk

lecithin and cholesterol were all added into ethanol

and mixed thoroughly until all the agents were

solute. The mixture was then dropped into to a

phosphate buffer solution maintained at 45℃ under

stirring. After 20 mins, the sample was subjected to

rotary evaporation and then sonication treatment.

The obtained samples were stored in refrigerator at

4 ℃ until further use. The nothing loaded liposomes

(control) and CSO loaded liposomes were also

prepared by the same procedure for comparison.

2.3 Particle Size Distribution

The particle size of all liposomal formations were

analyzed on a zeta sizer instrument (Nano ZS90,

Malvern Instruments, Malvern, UK)

2.4 Measurement of Lipid Oxidation

The extent of lipid oxidation in liposomes was

determined by an assay (thiobarbituric acid reactive

substances, TBARS) as described by Walker et al.,

2017 (

Walker, 2017)

. Briefly, 1 mL of liposomes

was added into 5 ml of TBA working solution,

mixed thoroughly and then heated in a water bath at

75℃ for 15 min. This mixture was cooled to room

temperature, and then centrifuged at 2500 rpm for

5min. The absorbance of supernatant at 532 nm was

recorded on a UV-visible spectrophotometer. The

TBARS content was calculated and expressed as ng

MDA equivalent per mg phospholipids.

2.5 UV-light Exposure Stability

In order to accelerate the instability induced by

UV-light irradiation, APPH was added into all

liposomes systems (Pires, 2019). Freshly prepared

liposomes added with APPH (0.05 mol/L) were

placed in closed quartz cells and irradiated with a

254 nm UVC germicide lamp (Philips TUV PL-S

5W/2P 1CT) at a radiance of 1.9W/m2. After

exposure for 15, 30, 45, 60, 90, 120 mins, samples

were withdrawn. The instability caused by UV

radiation was recorded by the determining size

distribution and MDA values.

2.6 Statistical Analysis

All determinations were repeated triplicate, and

presented as means ± standard deviations (SD). The

results were analyzed statistically for significance

(p≤ 0.05) using SPSS 18.0 software.

3 RESULTS & DISCUSSION

3.1 The Irradiation Stability of

Liposomes at 0.002% Antioxidant

Fig. 1A shows changes of the peroxidation product

in liposomes during irradiation. Obviously, the

MDA values of the control (nothing was loaded)

increased as the exposure time increased, indicating

oxidation of the liposomal bilayer occurred.

Meanwhile, the values of CSO loaded liposomes

were generally lower than that of the control

(p<0.05), suggesting the embedding CSO could

somewhat delay the oxidative degradation of the

membrane. This may be accounted to the antioxidant

activity of un-entrapped CSO. According to

literatures, the content of unsaturated fatty acid in

CSO was higher than 70% (Xiao, 2019). The

protective role of CSO may be originated from the

oxidization itself. In addition, the peroxidation

produced in liposomes containing antioxidants was

lower than that in CSO liposomes. What's more, the

MDA values of them was in the order of

THBQ>BHT>α-Tocopherol>β-carotene. The higher

antioxidant effect of β-carotene may be explained by

its conjugated polyenes structure, which could help

to remove free radicals and quench singlet oxygen,

as a result, inhibiting the decomposition of primary

oxidation products to secondary oxidation products

(

Walker,

2017). The result was consistent with our

previous report that the co-loaded CSO and β-

carotene exhibited synergistic antioxidant (Bai, 2019).

The Inﬂuences of Antioxidants on the Stability of Coix Seed Oil Liposomes Under Ultraviolet Irradiation

*indicates the value of the Control is significantly different from that of all the other liposomes at each time point (p<0.05).

Figure 1: The effects of antioxidant on oxidation stability of CSO liposomes during UV irradiation. A (0.002%), B (0.01%),

C (0.02%).

Figure 2 shows that particle size distribution of all

samples generally shifted to higher values after UV

irradiation. What’s more, the longer the exposure

time the larger the particle was. The results were in

consistence with previous reports (

Palmina, 2021;

Pires, 2019). Pires et al. (Pires, 2019) found that UV

irradiation affects the phosphate and carbonyl

groups of 1, 2-dipalmitoyl-sn-glycero-3-

[phospho-rac-(1-glycerol) in liposome. The enlarged

particle size may be due to the oxidation of

phospholipids induced by UV irradiation, which

might change the emulsifying property of

phospholipids, and disturbing the integrity and

stability of liposomes in the end. Meanwhile, more

encapsulated CSO may have leaked out from

liposomes and absorbed on the surface of bilayers as

the result of the changed integrity, that the increased

viscosity may promote particle aggregation (Sabet,

2021). In addition, the particle size changes for

liposomes containing CSO+BHT was the smallest,

indicating incorporating BHT into liposomes could

supply better shield against UV radiation.

FSB 2022 - The International Conference on Food Science and Biotechnology

Figure 2: The effects of 0.002% antioxidant on the particle size distribution of liposomes during irradiation. A:

L(CSO+β-carotene), B: L(CSO+TBHQ), C: L(CSO+BHT), D: L (CSO+α- tocopherol).

3.2 The Irradiation Stability of

Liposomes at 0.01% Antioxidant

Similarly, the MDA value were all increased as the

function of time, and the values were in the order of

control>CSO liposomes>CSO + antioxidant

liposomes at fixed irradiation time when 0.01%

antioxidant was embedded (Fig. 1B). It indicates

that all types of antioxidant exhibited certain

inhibition effect on the degradation of liposomes

(Temprana, 2011). In addition, comparing with Fig.

1A, the MDA values increased more slowly,

indicating fewer lipids were decomposed and the

anti-oxidative activity of antioxidant were increased

when the concentration of antioxidant increased

(Feng, 2018). However, nearly no significant

difference was detected among the liposomes loaded

with antioxidant.

Figure 3 shows that the particle size distribution

of the liposomes added with BHT or TBHQ

exhibited slightly shifts during irradiation, while that

of ones enclosed with α-tocopherol or β-carotene

was more complex. This suggests that BHT and

TBHQ at higher concentration could supply good

protection against the size change induced by

UV-light. It could be found that, the size of CSO+α-

tocopherol co-loaded liposomes became larger after

exposure for 45 mins, whereas, the size changed to

much smaller after 90 mins’ irradiation. According

to literatures, there are continuous movement and

collision among liposomal particles (Brilliantov,

2007). What’s more, the leakage of embedded

materials and state transition of bilayer membranes

co-existed during irradiation and affected the

collision (Pires, 2019). When the irradiated time was

short, α-tocopherol could supply efficient protection

for phospholipids, resulting in low extent of

oxidation. At this stage, the increase in particle size

caused by the collision may be dominantly

influenced by the leakage of embedded materials,

which enhanced the surface viscosity of liposomes

and increased the chances adhering with each other.

However, a large part of unsaturated fatty acids

might have undergone peroxidation during

long-term irradiation since the limited ant oxidative

activity of α-tocopherol. As a result, the melting

point of phospholipid bilayer membrane was

increased and transferred to gel phase, leading to

increased rigidity with decreased deformability for

liposomal membrane, which in turn inhibited the

leakage of CSO.

3.3 The Irradiation Stability of

Lipidosomes at 0.02% Antioxidants

Fig. 1C showed that the general trends of all

samples at 0.02% antioxidants were increased as

increasing the irradiation time. What’s more, the

The Inﬂuences of Antioxidants on the Stability of Coix Seed Oil Liposomes Under Ultraviolet Irradiation

Figure 3: The effects of 0.01% antioxidant on the particle size distribution of liposomes during irradiation. A:

L(CSO+β-carotene), B: L(CSO+TBHQ), C: L(CSO+BHT), D: L (CSO+α- tocopherol).

Figure 4: The effects of 0.02% antioxidant on the particle size distribution of liposomes during irradiation. A:

L(CSO+β-carotene), B: L(CSO+TBHQ), C: L(CSO+BHT), D: L (CSO+α- tocopherol).

MDA value was lower than that with less

antioxidant, except for the one with β-carotene.

Obviously, the MDA value of liposomes containing

CSO+ β-carotene was higher than that containing

less β-carotene after irradiation for the same time.

What’s more, some values were even higher than

that of CSO liposomes. This suggests that

β-carotene might exhibit good antioxidative at

FSB 2022 - The International Conference on Food Science and Biotechnology

limited concentration, whereas would promote

oxidation when excess certain concentration.

Scoccianti et al. reported tocopherols could help

against lipid peroxidation induced by 1 mM Cr(III),

but generated oxidative stress at the highest

concentration (Scoccianti, 2016).

Figure 4A shows that there are significant

changes in particle size distribution of L

(CSO+β-carotene) during irradiation. And the mean

particle size was in the order of 90 mins treated > 45

mins treated > freshly prepared, indicating

irradiation induced polymerization of the liposome.

As to L (CSO+α- tocopherol), the mean particle size

of 90 mins treated was smallest, while 45 mins

treated samples were the largest. On the contrary, the

changes in L (CSO+TBHQ), L (CSO+BHT) were

not so significant, indicating that TBHQ and BHT

had a strong irradiation stabilization effect at this

concentration.

4 CONCLUSIONS

In this research, the influence of TBHQ, BHT, α-

Tocopherol and β-carotene on the physiochemical

stability of CSO liposomes during UV irradiation

were investigated. The results showed that CSO

could exert certain protection for liposomal bilayer

from oxidation. The ant oxidative efficiency of

antioxidant was largely dependent on the type,

concentrations, and exposure time. β -carotene could

supply good shield at low concentration, whereas

promote oxidation when the concentration increased

to 0.2%. On the contrary, TBHQ and BHT exhibited

good irradiation stabilization effect, and nearly no

particle size changes were detected for all

concentrations. Although the oxidation of CSO

liposomes could be inhibited by α- tocopherol; its

particle size stabilization function was limited.

However, whether these antioxidants influence the

leakage of CSO and the structural integrity of

liposomal bilayer need further investigation.

ACKNOWLEDGMENTS

This research was funded by the Jiangxi Provincial

Department of Education Project (Grant number

GJJ201114), National Natural Science Funds of

China (Grant number 31560465), and Open fund

project of Jiangxi Aquatic Product Processing and

Safety Control Engineering Research Center (Grant

number KFJJ2101, KFJJ2102).

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