The preparation and antioxidant activities of three phenyl-acylchitooligosaccharides

Keywords: Chitooligosaccharide, Phenyl-acylchitooligosaccharide, Antioxidant activity, Radical scavenging effect

3.1. Materials

Low molecular weight chitosan (LCS) is a commercial material provided by Qingdao Yunzhou Biotechnology Co., Ltd. (China). It's deacetylation degree >90%, average molecular weight (Mw) of 3 kDa. Chitooligosaccharide (COS) with a molecular weight of 1.1 kDa was prepared in our laboratory by the combined degradation of microwave and acetic acid. Tris (Tris), Reduced Coenzyme I (NADH), Safranine O, Nitrotetrazolium Blue (NBT), Phenazine Potassium Sulfate (PMS), 1,1-Diphenyl-2-Trinitrophenylhydrazine (DPPH) was purchased from Sigma Chemicals Co. Disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, ferrous sulfate, disodium ethylenediaminetetraacetate (EDTA), absolute ethanol, 30% hydrogen peroxide (H₂O₂), 1-ethyl-(3 -Dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS), 2-morpholinoethanesulfonic acid (MES), salicylic acid (BHA), α-naphthylacetic acid (NAA), indole-3-butyric acid (IBA). All other chemicals and reagents were of analytical grade and were used without further purification.

3.2. Analytical methods

The infrared spectra of COS and its derivatives were detected using a Spectrum 65 (PerkinElmer) Fourier-Transform Infrared (FTIR) Spectrometer (Inner Mongolia Agricultural University, Hohhot, China) ranging from 4000 to 400 cm⁻¹ at 25 °C. The ¹³C nuclear magnetic resonance (NMR) spectra were recorded at room temperature using a “MAS”-Magic Angle Spinning model test (Qingdao Xinfei Testing Co., Ltd., Qingdao, China). Elemental analyses were performed on a Leeman EA3000 elemental analyzer Inner Mongolia Agricultural University. All the samples were weighed using a Mettler XP6 millionth balance. The sample weights ranged from 1.5 to 2.5 mg. The degrees of substitution (DS) were calculated in accordance with the carbon to nitrogen (C/N) ratios of COS and its derivatives. The DS of chitooligosaccharide derivatives were estimated employing the following formula [17, 18]:

DS = m(C/N)1−(C/N)0n, where (C/N)₁ is the C/N of the modified COS, (C/N)₀ is the C/N of the original COS, and m and n are the numbers of nitrogen and carbon atoms, respectively, introduced by the COS modification.

3.3. Preparation of phenyl-acylchitooligosaccharide

The molar ratio of containing benzene carboxylic acid and chitooligosaccharide was set as 20:1 and 7:1 with reference to the polymerization degree of chitooligosaccharide. The containing benzene carboxylic acid was dissolved in MES- ethanol buffer solution (pH = 5.5) with stirring until complete dissolution. EDC·HCl and NHS were added to continue stirring for 3 h to activate the carboxyl group (the molar ratio of containing benzene carboxylic acid: EDC·HCl: NHS = 1:3:3). Then, chitooligosaccharide was added and stirred at room temperature for 24 h. The solution was dialyzed against distilled water for 48 h using a 500 Da MW cut-off dialysis membrane. Then rotate the solution in the dialysis bag to 30–50 mL (the concentration temperature is controlled below 55 °C). Finally, the concentrated solution was freeze-dried for more than 48 h to obtain a tawny or brown target product powder.

3.4. Hydroxyl radical assay

The hydroxyl radicals scavenging ability of COS and phenyl-acylchitooligosaccharide were assessed by the method of Zhong et al. [19]. The scavenging ability was calculated as follows:

E% = (Asample−AblankAcontrol)×100; among them, the A_sample is absorbance value of the experimental sample; the A_control is absorbance value of the reaction system without sample; the A_blank is absorbance value of distilled water as blank group.

3.5. Superoxide radical scavenging assay

The superoxide radicals scavenging ability of COS and phenyl-acylchitooligosaccharide were assessed by the method of Zhong et al. [20]. The capability of scavenging superoxide radical was calculated using the following equation:

E% = (1−AsampleAcontrol)×100; among them, the A_sample is absorbance value of the experimental sample; the A_control is absorbance value of the reaction system without sample.

3.6. Measurement of reducing power

The reducing power of COS and phenyl-acylchitooligosaccharide was assessed by the method of Zhong et al. [20]. The increase in absorbance indicates that the reduction ability is enhanced.

3.7. 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) radical scavenging ability

The DPPH radical scavenging ability of COS and phenyl-acylchitooligosaccharide was assessed by the method of Curcio et al. [21]. The capability of scavenging DPPH radical was calculated using the following equation:

E% = 1−[(A1−A2A0)×100)]; among them, the A₁ is absorbance value of the first measurement; the A₂ is absorbance value of the second measurement; the A₀ is absorbance value of the reaction system without sample.

3.8. Statistical analysis

All data are expressed as means ± SD. Data were analyzed by an analysis of variance (P < 0.05) and the means separated by Dun-can's multiple range tests. The results were processed by computer programs: Excel, GraphPad Prism 5 and Origin 2021.

4.1. Structure and physicochemical characteristics of the derivatives

Figure 1 presents the comparison of transmission FT-IR spectra data for phenyl-acylchitooligosaccharide with original chitooligosaccharide at different molecular weights. According to Figure 1, the characteristic broad peaks of stretching vibration of O–H and N–H located at 3500-3200 cm⁻¹ are obviously shifted, indicating that N–H may react. The broad peak generated by the associated –OH stretching vibration is the most important feature of carboxylic acid, which is also the best proof for the acylation reaction between the target group and the chitooligosaccharide. The characteristic absorption peak of –NH₂ at 1500–1510 cm⁻¹ (namely the band of amide Ⅱ) is enhanced, indicating that –NH₂ has undergone acylation reaction. The characteristic absorption peak of C=O stretching vibration (namely the amide Ⅰ band) appears at 1640–1650 cm⁻¹. And the C=C skeleton stretching vibration of aromatic ring is also concentrated in this region. An obvious saturated –CH stretching vibration absorption peak appears at 2980–2940 cm⁻¹. Located at 1210–1380 cm⁻¹ are the absorption peaks of –CH₂ rocking vibration and asymmetric C2 deformation. The out-of-plane bending vibration of C–H on the aromatic ring is absorbed at 900–650 cm⁻¹. The position of the absorption peak in this region has nothing to do with the nature of the substituents on the aromatic ring, but is related to the number of hydrogens connected to the aromatic ring. The lower the vibration frequency, the greater the absorption intensity. Located at 761 cm⁻¹ is the characteristic absorption peak of ortho-disubstituted benzene ring. The characteristic absorption peaks of naphthalene ring are located at 780 cm⁻¹ and 651 cm⁻¹. Located at 655 cm⁻¹ is the characteristic absorption peak of indole ring. All the above results indicated that three kinds of phenyl-acylchitooligosaccharide were obtained.

Figure 2 show the ¹³C NMR spectra of o-hydroxybenzoyl-chitooligosaccharide (BHACS), α-naphthylacetyl-chitooligosaccharide (NAACS), and 3-Indolebutyryl-chitooligosaccharide (IBACS), respectively. According to the display of (a), at 174.61 ppm is the chemical shift of the C=O carbon of the amide bond. Located at 98.02(C1), 76.42(C4), 74.83(C5), 72.00(C3), 60.39(C6), 55.37(C2) ppm are the chemical shifts of the CS ring. Located at 116.43–160.53 ppm are the chemical shifts of the benzene ring skeleton of BHA. According to the display of (b), at 174.77 ppm is the chemical shift of the C=O carbon of the amide bond. Located at 98.05(C1), 76.43(C4), 74.84(C5), 72.11(C3), 61.83(C6), 55.38(C2) ppm are the chemical shifts of the CS ring. Located at 133.61–160.61 ppm are the chemical shifts of the naphthalene ring skeleton of NAA. According to the display of (c), at 174.62 ppm is the chemical shift of the C=O carbon of the amide bond. Located at 98.07(C1), 76.44(C4), 74.26(C5), 72.04(C3), 60.40(C6), 55.38(C2) ppm are the chemical shifts of the CS ring. Located at 102.22–160.55 ppm are the chemical shifts of the indole ring skeleton of IBA. All the above results indicated that three kinds of phenyl-acylchitooligosaccharide were obtained.

The results of elemental analyses, yield and grafted degree of the derivatives are listed in Table 1. The yields of BHACS, NAACS and IBACS were 46.84%, 41.11% and 49.18%, respectively. The elemental analyses indicate that the substitution degree of BHACS, NAACS, and IBACS about 74.47%, 28.62% and 71.12% respectively.

4.2. Hydroxyl radical scavenging activity of chitooligosaccharide and phenyl-acylchitooligosaccharide

Hydroxyl radicals are generated by the EDTANa₂-Fe (II)–H₂O₂ system. Safranin O can be uniquely targeted by hydroxyl radicals, causing it to fade. The content of hydroxyl radicals can be measured by colorimetry according to the degree of fading [22]. The scavenging ability of chitooligosaccharide and phenyl-acylchitooligosaccharide was determined based on this method. The results are plotted in Figure 3.

As shown in Figure 3, all derivatives have scavenging activity. The scavenging ability of the derivatives increased with increasing of the concentration. The highest clearance rates of derivatives in (a), (b), (c) were 11.43%, 8.29%, and 4.98%, respectively. Taking Vc as a reference, the IC50 values of all derivatives were greater than Vc. The IC50 value of BHALCS was 2.812 mg/mL, which was the best scavenging ability among the derivatives. For BHACS, NAACS and IBACS, the clearance rates of BHALCS, NAALCS and IBALCS were higher than those of BHACOS, NAACOS and IBACOS as a whole. This is because the high molecular weight derivatives have longer molecular chains, more connected groups, and more active hydrogens, which can react with more hydroxyl radicals to achieve the purpose of scavenging. However, because the low molecular weight derivatives have short molecular chains and also have many active hydrogens, the difference in the scavenging abilities of the two derivatives with different molecular weights is small. Moreover, the scavenging abilities of these three derivatives were lower than those of the three starting materials. This may be because the acylation reaction makes the amino group on the chitooligosaccharide connect with the carboxyl group of the carboxylic acid, thus losing a lot of active hydrogen. Even the scavenging ability of the derivative is lower than that of the raw material itself. All the results showed that the scavenging ability of chitooligosaccharide grafted containing benzene carboxylic acid was not significant, which needs further study.

4.3. Superoxide radical scavenging activity of chitooligosaccharide and phenyl-acylchitooligosaccharide

The main cause of oxygen toxicity in the body is due to the toxic effect of superoxide anion [23]. The results of superoxide anion scavenging with chitooligosaccharide and phenyl-acylchitooligosaccharide are shown in Figure 4.

The inhibitory effects of various phenyl-acylchitooligosaccharide on superoxide anion free radicals were significant and enhanced with the increase of concentration. The highest clearance rates of derivatives in (a), (b), (c) were 32.13%, 47.44%, and 36.92%, respectively. For superoxide anion scavenging ability, both NAALCS and NAACOS were outstanding with IC50 values of 0.542 mg/mL and 0.546 mg/mL, respectively. The inhibitory ability of BHACOS, NAACOS and IBACOS was stronger than that of BHALCS, NAALCS and IBALCS. The removal of superoxide anion is mainly due to the hydrogen atom transfer mechanism, that is, the hydrogen atoms shed from the antioxidant are transferred to O₂·⁻. The number of free hydroxyl groups on the end groups of low molecular weight chitooligosaccharides is higher than that of high molecular weight chitooligosaccharides. And the introduction of BHA, NAA and IBA increases the number of active hydrogen atom. The hydrogen atoms on the O–H bond and N–H bond are shed and transferred to O₂·⁻, and the negative charge on O₂·⁻ is transferred to the position where the hydrogen atoms are shed. To achieve free radical scavenging effect. However, the reason for the high scavenging ability of NAALCS and NAACOS is probably not due to the grafting of NAA. The degree of substitution of NAA is low, and most of the scavenging ability of its derivatives is derived from chitooligosaccharide itself.

4.4. Reducing power of chitooligosaccharide and phenyl-acylchitooligosaccharide

The reducing power is usually associated with the presence of reductones, which act as antioxidants by donating a hydrogen atom to disrupt the free radical chain. Figure 5 show the reducing ability of various phenyl-acyl-chitooligosaccharides.

The reducing ability of all derivatives showed a good linear relationship with concentration. The absorbance values in (a), (b), and (c) are all low, and the highest value is only 0.032 Abs. But the reducing ability of BHACS, NAACS and IBACS is very weak. The reducing power of no derivative can be higher than that of the chitosan oligosaccharide raw material. This is probably because the acylation reaction caused a large amount of damage to the reductone structure in the molecule, and then lost the reducing ability. Further research is needed on how to improve the reduction ability.

4.5. DPPH radical scavenging activity of chitooligosaccharide and phenyl-acylchitooligosaccharide

DPPH is 1,1-diphenyl-2-picrylhydrazino radical. There are multiple electron-withdrawing –NO₂ and benzene ring large π bonds in the molecule. It is an ideal pharmacological model for the detection of free radical scavenging activity [24]. Figure 6 show the scavenging ability of various phenyl-acylchitooligosaccharide.

The scavenging ability of all derivatives is linear with concentration. The highest clearance rates of derivatives in (a), (b), (c) were 80.27%, 34.93%, and 77.66%, respectively. The BHALCS and IBALCS were significantly effective with IC50 values of 0.263 mg/mL and 0.253 mg/mL, respectively; these were lower than the IC50 values of BHA and IBA, 6.596 mg/mL and 0.663 mg/mL. The scavenging ability of BHACS and IBACS was higher than that of chitooligosaccharide raw materials. The scavenging ability of NAACS is also limited by the low degree of substitution. The reaction system of the DPPH free radical scavenging experiment was anhydrous ethanol. The solubility of chitooligosaccharide in absolute ethanol is very weak. However, the groups of derivatives (salicylic acid, α-naphthylacetic acid, indole-3-butyric acid) have very high solubility in ethanol. This makes the scavenging effect of the derivatives on DPPH free radicals better than the above three tests. Some effects of different test system environments on the solubility and polarity of derivatives will indirectly interfere with the release of active hydrogen.

Author contribution statement

Yao Liu: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Fang Wen, Hehe Yang: Performed the experiments; Analyzed and interpreted the data.

Liangliang Bao: Analyzed and interpreted the data.

Zhihong Zhao: Contributed reagents, materials, analysis tools or data.

Zhimei Zhong: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Funding statement

This work was supported by Inner Mongolia Science and Technology Project (2021GG0078) and Inner Mongolia Major Science and Technology Project (2019ZD001).

Data availability statement

Data included in article/supplementary material/referenced in article.

Declaration of interest’s statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.

• 1.Pu T.L., Han X.Q., Den H.S. Research progress of extraction method of antioxidant components and antioxidant activity of moringa oleifera. Sci. Technol. Food Ind. 2019;40:310–315. [Google Scholar]
• 2.Abeyrathne E., Huang X., Ahn D.U. Antioxidant, angiotensin-converting enzyme inhibitory activity and other functional properties of egg white proteins and their derived peptides-A review. Poultry Sci. 2018;97:1462–1468. doi: 10.3382/ps/pex399. [DOI] [PubMed] [Google Scholar]
• 3.Wang Y., Wang X.H., Xu B.Z. Research progress of metallothionein function in vivo antioxidant. Sci. Technol. Food Ind. 2016;37:377–385. [Google Scholar]
• 4.Horton J.W. Free radicals and lipid peroxidation mediated injury in burn trauma: the role of antioxidant therapy. Toxicology. 2003;189:75–88. doi: 10.1016/s0300-483x(03)00154-9. [DOI] [PubMed] [Google Scholar]
• 5.Zhao Z.Q., Wang M., Zhang Z.Q. Research progress of antioxidation efficacy and extraction of plant polysaccharide. Sci. Technol. Food Ind. 2018;39:337–342. [Google Scholar]
• 6.An C.Y., Qi Z.M., Su X.L. Research advance in Terminalia antioxidants. Chem. Life. 2020;40:925–932. [Google Scholar]
• 7.Liu H., Ma S.H., Li C.H. Latest application progress of infrared spectroscop in the researchon the antioxidant of plants. Spectrosc. Spectr. Anal. 2016;36:91–92. [Google Scholar]
• 8.Liu Y.T., Li J.L. Advances in research on antioxidant activity of polysaccharides in vitro. Food Res. Dev. 2019;40:214–219. [Google Scholar]
• 9.Liu J., Jia P.Y., Jiao S.M. Comparison of biological activity of three common chitosan oligosaccharide salts. J. Northwest A&F University (Nat. Sci. Ed.). 2019;47:139–144. [Google Scholar]
• 10.Chae S.Y., Son S., Lee M. Deoxycholic acid-conjugated chitosan oligosaccharide nanoparticles for efficient gene carrier. Cont. Release. 2005;109:330–344. doi: 10.1016/j.jconrel.2005.09.040. [DOI] [PubMed] [Google Scholar]
• 11.Einbu A., Varum K.M. Depolymerization and denacetylation of chitin oligomers in hydrochloric acid. Biomacromolecules. 2007;8:309–314. doi: 10.1021/bm0608535. [DOI] [PubMed] [Google Scholar]
• 12.Lodhi G., Kim Y.S., Hwang J.W. Chitooligosaccharide and its derivatives: preparation and biological applications. BioMed Res. Int. 2014;2014 doi: 10.1155/2014/654913. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 13.Xu Q., Chen L.H., Qin Z. Monomer preparation and structure analysis of chitobiose and chitotriose. Sci. Technol. Food Ind. 2017;38:13–18. [Google Scholar]
• 14.Zou P., Yang X., Wang J. Advances in characterisation and biological activities of chitosan and chitosan oligosaccharides. Food Chem. 2016;190:1174–1181. doi: 10.1016/j.foodchem.2015.06.076. [DOI] [PubMed] [Google Scholar]
• 15.Basma Morad Mohamed Ali, Farouk Gheida Shereen, Nageh Ahmed El Mahdy, Nashaat Sadek Shery. Evaluation of salicylic acid peeling in comparison with topical tretinoin in the treatment of postinflammatory hyperpigmentation. J. Cosmet. Dermatol. 2017;16:12–17. doi: 10.1111/jocd.12301. [DOI] [PubMed] [Google Scholar]
• 16.Xu J.J., Xing Z., Sun J.Y. Effect of α-naphthylacetic acid on the cutting of Impatiens lingzhiensis. J. Plateau Agri. 2022;6:67–72. [Google Scholar]
• 17.Liu W.X., Qin Y.K., Liu S., Xing R.E., Yu H.H., Chen X.L., Li K.C., Li P.C. Ccoordinated O-carboxymethyl chitosan metal complexes: synthesis, characterization and antifungal efficacy. Int. J. Biol. Macromol. 2018;106:68–77. doi: 10.1016/j.ijbiomac.2017.07.176. [DOI] [PubMed] [Google Scholar]
• 18.Justi K.C., Favere V.T., Laranjeira M.C.M., Neves A., Casellato A. Synthesis and characterization of modified chitosan through immobilization of complexing agents. Macromol. Symp. 2005;229:203–207. [Google Scholar]
• 19.Zhong Z.M., Ji X., Xing R.E. The preparation and antioxidant activity of the sulfanilamide derivatives of chitosan and chitosan sulfates. Bioorg. Med. Chem. 2007;15:3775–3782. doi: 10.1016/j.bmc.2007.03.036. [DOI] [PubMed] [Google Scholar]
• 20.Zhong Z.M., Xing R.E., Liu S. The antioxidant activity of 2-(4(or 2)-hydroxyl-5-chloride-1,3-benzene-di-sulfanimide)-chitosan. Eur. J. Med. Chem. 2008;43:2171–2177. doi: 10.1016/j.ejmech.2007.10.018. [DOI] [PubMed] [Google Scholar]
• 21.Curcio M., Puoci F., Iemma F. Covalent insertion of antioxidant molecules on Chitosan by a free radical grafting procedure. J. Agric. Food Chem. 2009;57:5933–5938. doi: 10.1021/jf900778u. [DOI] [PubMed] [Google Scholar]
• 22.Jin J.Q., Ding D.N., Dong H.Y. The study of chemical and scavenging action to hydroxyl free radical of polysaccharides of Ginkgo biloba leaf. J. Xi'an Med. Univ. 2000;21:417–419. [Google Scholar]
• 23.Yan S., Fan Y.F., Zheng L.H. The extract from camellia pollen by Lactobacillus fermenting scavenging four different free radicals. Food Sci. Technol. 2017;42:238–243. [Google Scholar]
• 24.Li X.C., Lin J., Gao Y.X. Antioxidant activity and mechanism of rhizoma cimicifugae. Chem. Cent. J. 2012;6:140. doi: 10.1186/1752-153X-6-140. [DOI] [PMC free article] [PubMed] [Google Scholar]

Data Availability Statement

Data included in article/supplementary material/referenced in article.