Structure characteristics of low molecular weight pectic polysaccharide and its anti-aging capability by modulating the intestinal homeostasis

Structure characteristics o, low molecular weight pectic polysaccharide and its anti-aging capability by modulating the intestinal homeostasis(1).pdfStructure characteristics of low molecular weight pectic polysaccharide and

its anti-aging capability by modulating the intestinal homeostasis

Junhui Li a,b,1

, Lu Wang a,1

, Kun Yang a,1

, Guocai Zhang a,1

, Shan Li c

, Hongjian Gong d

,

Mingqi Liu a

, Xianjun Dai a,*

a College of Life Sciences, China Jiliang University, Hangzhou, Zhejiang 310018, China b Shandong (Linyi) Institute of Modern Agriculture, Zhejiang University, Linyi, China c Institute of Nutrition and Health, Qingdao University, Qingdao 266003, China d Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430016, China

ARTICLE INFO

Keywords:

Citrus peel

Pectic polysaccharide

Structure analysis

Anti-aging activity

Intestinal homeostasis

ABSTRACT

Pectic polysaccharide has attracted increasing attention for their potential biological properties and applications

in health industries. In this study, a low-molecular-weight pectic polysaccharide, POS4, was obtained from citrus

peel. The structure of POS4 was preliminarily analyzed by gel-permeation chromatography, monosaccharide

analysis, infrared spectroscopy (IR) and nuclear magnetic resonance spectroscopy (NMR). Results showed that

the molecular weight of POS4 was 4.76 kDa and it was a galacturonic acid enriched pectic polysaccharide. The

anti-aging activity in vivo showed that POS4 could notably prolong the average lifespan of fruit flies by sup￾pressing the generation of reactive oxygen species (ROS). Further studies demonstrated that POS4 could enhance

intestinal homeostasis by modulating gut microbiota in a positive way and regulating autophagy associated

genes. Taken together, we proposed that galacturonic acid enriched low molecular weight pectic polysaccharide

have great potential in the development of healthy foods such as anti-aging health care products.

1. Introduction

Aging is characterized by progressive decline in physiological func￾tions accompanied by an accumulating risk of age-associated diseases

(Lopez-Otin et al., 2013). Recently, an increasing number of researches

have highlighted the pivotal roles of the intestines in aging and lifespan

modulation (Fan et al., 2018; Funk et al., 2020). The intestinal epithe￾lium functions as a barrier to the environment and acts as an integration

site to respond to diverse intrinsic as well as extrinsic stimuli. Studies

demonstrated that promoting intestinal homeostasis and reducing

epithelial barrier dysfunction contributes to lifespan extension (Hu &

Jasper, 2019; Salazar et al., 2018). Furthermore, autophagy and

oxidative damage induced by ROS is presumed to be critical roles in the

aging process (Beckman & Ames, 1998; Funk et al., 2020). Recent

studies revealed that intrinsic autophagy could lead to DNA damage

clearance, renewing stem cell activity, maintaining intestinal homeo￾stasis, thus retarding tissue aging and prolonging animal lifespan. For

ROS, its formation keeps stable or increases with the growth of age, but

the antioxidant defense ability of the body gradually declines, giving rise

to the redox imbalance as a key pathogenic factor in numerous age￾related disorders including cancer, Alzheimer's disease and cardiovas￾cular disease (Jackson & McArdle, 2016). Therefore, improved intesti￾nal homeostasis and enhanced ROS scavenging has become a central

research emphasis of prompting health and retarding aging (Finkel &

Holbrook, 2000).

With the rapidly increasing of aged population and the pressure of

social support for the aged, a soaring demand for healthy aging has

received much attention from clinical, social and fiscal perspectives

(Feng et al., 2015). It has been demonstrated that high-calorie intake,

malnutrition, and oxidative stress exhibit negative effect on the lifespan

and induce chronic diseases (Santos-Lopez et al., 2017). Hence, dietary

functional ingredients with significant anti-aging activity are in great

demand. Polysaccharides are biopolymers consisting of mono￾saccharides linked together through glycosidic bonds. Accumulating

evidences have documented that polysaccharide from natural sources

can improve intestinal barrier function by modulating gut microbiota

and related genes and protect the organisms from damage caused by free

radicals with negligible adverse effects, indicating the great potential of

* Corresponding author.

E-mail address: xjdai@cjlu.edu.cn (X. Dai). 1 These authors contributed equally to this work.

Contents lists available at ScienceDirect

Carbohydrate Polymers

journal homepage: www.elsevier.com/locate/carbpol

https://doi.org/10.1016/j.carbpol.2022.120467

Received 13 June 2022; Received in revised form 28 November 2022; Accepted 11 December 2022

Carbohydrate Polymers 303 (2023) 120467

2

polysaccharides as anti-aging agent (Jin et al., 2017; Li et al., 2017).

Pectic polysaccharides are a broad class of exopolysaccharides present in

plant cell walls. As a nature fiber in plant, pectic polysaccharides

increasingly gain attention as a health promoting polysaccharide and

possess broad range of pharmacologic activity, such as antioxidant ac￾tivity, antitumor, prebiotic and immunomodulatory activity (Lee et al.,

2022; Lv et al., 2020; Zou et al., 2020). Low molecular weight pectic

polysaccharides are obtained by the degradation of pectic poly￾saccharides (Gullon et al., 2013). In comparison with pectic poly￾saccharide, pectic oligosaccharide exhibits better solubility and

improved bioavailability (Kapoor & Dharmesh, 2017; Li, Li, Zheng,

et al., 2019). Therefore, the low molecular weight pectic oligosaccharide

is currently of great interest (Li, Li, Liu, et al., 2019). It has been reported

that despite the benefit of pectic oligosaccharide to age-associated dis￾eases including cancer, diabetes and cardiovascular, its anti-aging effect

and the underlying mechanisms at the organismal level remain

enigmatic.

Based on the mentioned characteristics above, we hypothesized that

low-molecular-weight pectic polysaccharide could exhibit anti-aging

effect through scavenging ROS and promoting the intestinal homeosta￾sis. To test the possibility, POS4, a low molecular weight pectic poly￾saccharide from by-products of citrus processing industry was prepared

by Superdex 75 gel filtration chromatograph and the structure proper￾ties of POS4 were characterized by FT-IR, NMR spectroscopy and

monosaccharide composition analysis. D. melanogaster is a useful and

appropriate model in aging research for their short lifespan, easy

maintenance, and the fully sequenced genome. Therefore, the effect of

POS4 on lifespan extension in Drosophila was determined and the un￾derlying anti-aging mechanism of POS4 was further explored by eval￾uating the antioxidant activity and intestinal homeostasis. This study

will contribute to the exploration of such pectic polysaccharide com￾ponents in the development of useful pro-longevity pharmaceuticals and

also bring great economic and social benefits to the society.

2. Materials and methods

2.1. Materials

Ultrahydrogel 250 and Superdex 75 gel filtration chromatograph

was from Waters (Milford, USA) and GE Healthcare (Marlborough,

USA). Deuterium oxide and other chemical reagents were obtained from

Aladdin Chemical Reagent Co., Ltd.

2.2. Preparation of low molecular weight pectic oligosaccharides

The low molecular weight pectic polysaccharide (LMWP) (Product

number: GYKP002) extracted from mandarin citrus peel is a commercial

product and kindly donated as gift from Zhejiang Gold Kropn Biotech￾nology Co., Ltd. The LMWP was dissolved in distilled H2O and frac￾tionated by a Superdex 75 10/300 GL column eluted with 0.3 M

NH4HCO3. Carbohydrate fragments were gathered every 3 min with a

test tube and the polysaccharide content was detected with the phenol￾sulfate colorimetry (Chen et al., 2011).

2.3. The molecular weights (Mw) analysis of POS4

The Mw of POS4 was analyzed by high performance size exclusion

chromatography with refractive index detector (Li, Li, et al., 2016). The

Ultrahydrogel 250 column (Waters, Milford, USA) was used for the

separation of POS4. The sample was eluted by 0.2 M NaCl at a flow rate

of 0.5 mL/min. Dextran standards were used to obtain standard curve to

determine the molecular weight of POS4.

2.4. Monosaccharide composition and linkage determination

Monosaccharide composition of POS4 was determined by the 1-

phenyl-3-methyl-5-pyrazolone (PMP) high performance liquid chroma￾tography (HPLC) method (Li, Li, Zheng, et al., 2019). Briefly, 2 mg of

POS4 was treated with 4 M trifluoroacetic acid (TFA) at 110 ◦C for 8 h.

Then anhydrous methanol was used and dried in drying oven to remove

TFA and the sample solution was adjusted to the alkaline with NaOH

solution. The hydrolysate was derivatized with 0.5 M PMP solution at

70 ◦C for 30 min in alkaline condition. After the completeness of deri￾vation of POS4, chloroform was used to obtain the PMP derivatives

which were analyzed by a ZORBAX Eclipse XDB-C18 column (Agilent, 5

μm, 4.6 mm × 250 mm).

The methylation analysis was conducted to determine the glycosidic

linkage of

Carbohydrate Polymers 303 (2023) 120467

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(TaKaRa), and Nanodrop ND-1000 and formaldehyde gel electropho￾resis was applied to judge the quality and quantity of RNA. Cdna was

synthesized using the Thermoscript real-time PCR kit (Invitrogen)

following the manufacturer's protocols. Mrna expression was deter￾mined with the SYBR green Qpcr detection method and the relative

quantification for the expressed genes was estimated using the 2− ΔΔCt

method. The primer sequences used in the study are exhibited in Table 1.

2.15. Statistical analysis

The significance differences between the life spans of control and

POS4-treated fruit flies under normal condition were determined by

GraphPad Prism 6 software (GraphPad Software, La Jolla, CA, USA). (*)

p < 0.05 and (**) p < 0.01 indicate statistically significant differences

between the data.

3. Results and discussions

3.1. Isolation and structural analysis of POS4

3.1.1. General properties of POS4

The elution curve of Superdex 75 gel filtration chromatograph for

low molecular weight pectic polysaccharide was shown in the Fig. 1a,

from which four fractions of low molecular weight pectic polysaccharide

were analyzed by phenol-sulfuric acid method after chromatography.

The solution of 17–23 tubes, 32–36 tubes, 40–45 tubes, and 47–58 tubes

was collected followed by freeze drying to obtain fractions POS1 (yield

12.4 %), POS2 (yield 15.8), POS3 (yield 11.3 %) and POS4 (yield 60.5

%), respectively. Among them, POS4 is the most abundant component of

low molecular weight pectic polysaccharide. Therefore, POS4 was

selected and prepared for subsequent analysis.

The molecular weight of POS4 was calculated judging by high per￾formance gel permeation chromatography (HPGPC). As shown in the

Fig. 1b, POS4 exhibited a simple symmetric peak, indicating POS4 was a

homogeneous polysaccharide component. Judging by the standard

curve, the molecular weight of POS4 was calculated to be 4.76 ± 0.082

kDa.

Further chemical composition analysis (Fig. 2a) by PMP-HPLC

showed that POS4 was mainly consisted of Man, Rha, GlcA, GalA, Glc,

Gal, Xyl, Ara and Fuc. GalA was the enriched component in POS4. The

molar parts of HG and RG-I were investigated as follows: HG = GalA −

Rha, RG-I = [GalA − HG] + Rha + Ara + Gal (Shakhmatov et al., 2014).

According to relative proportions of HG to RG-I (77.2:15.6), the back￾bone of polysaccharide moiety in PSO4 is likely HG. Molar ratio of GalA

to Rha in POS4 was calculated as 19.21, a typical characteristic for HG,

suggesting that POS4 is mainly composed of 1, 4-D-galacturonicacid

(GalA). Moreover, the linkage types of POS4 were determined by

methylation analysis. As shown in Table 2, POS4 mainly contained six

monosaccharide residues (rhamnose (4.07 %), galacturonic acid (82.43

%), galactose (5.54 %), glucose (3.39 %) mannose (2.24 %) and arabi￾nose (2.33 %). The molar ratios of total non-reducing terminal residues,

total branching points and linear residues were 11.31 %, 10.38 % and

78.31 %, respectively, suggesting a relatively low branched structure in

POS4. In addition, the existence of 1,4-linked GalA and 1,2,4-linked Rha

indicated the presence of rhamnogalacturonan I region (RG-I) in POS4

and the high ratio of 4-GalpA (68.04 %) proved that HG was the

dominant structure in the main chain, in line with monosaccharide

composition analysis. It has been demonstrated that acid treatment

could break up the acid-labile linkages between the GalA and rhamnose

(Rha) residues in the RG-I region and induce the loss of neutral sugar

side-chains, generating low molecular weight pectic polysaccharide

with high proportions of HG (Harris & Smith, 2006), consistent with our

results.

3.1.2. IR spectroscopy and NMR analysis of POS4

The infrared spectrum of POS4 was illustrated in Fig. 2b. An intense

peak at around 3354 cm− 1 corresponds to stretching of hydroxyl groups

(OH) and the signals about 3453 cm− 1 is the characteristic C–H

stretching vibrations. The ratio of the peak area ascribed to carboxylic

ester over the sum of peak area assigned to carboxylic ester and

Fig. 2. The structure characteristics of POS4. (a) Monosaccharide composition analysis of POS4 by PMP-HPLC; (b) The FT-IR spectra of POS4.

Table 2

Methylation analysis results of POS4: sugar residues and molar ratios.

Deduced

residue

Linkage

patterns

RTa Mol

%b

Total monosaccharide

based

Rha4 2,4-Rhap 11.711 4.07 4.07

GalAt t-GalpA 9.299 11.31 82.43

GalA 4-GalpA 13.082 71.12

Gal 3-Galp 12.131 2.22 5.54

Gal 6-Galp 14.711 1.58

Gal4 3,4-Galp 15.002 1.74

Glc 3-Glcp 11.467 1.75 3.39

Glc 4-Glcp 13.344 1.64

Man4 2,4-Manp 16.168 2.24 2.24

Ara25 2,3,5-Araf 17.671 2.33 2.33

Letter labeled by a superscript t refers to a terminal residue; The superscript

numbers follow the letters indicate the branching sites in these residues; Data for

sugar residues <1 % are not shown. a RT: retention time is relative to 4-Glcp. b Mol (%): molar ratio of each sugar residue is based on the percentage of its

peak area.

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carboxylic acid groups has been calculated as the degree of methyl

esterification (DE) (Fellah et al., 2009). The peak at 1606 cm− 1 corre￾sponds to the C–

–O stretching vibration of ionic carboxyl groups and no

obvious signals attributed to carboxylic ester could be observed,

implying the non-esterified pectic polysaccharide. Moreover, the

absorption peaks between 1000 and 1150 cm− 1 were assigned to the

stretching vibrations C–OH side groups and the C–O–C glycosidic bond

vibration and bands at about 1074 and 1045 cm− 1 may be attributed to

galactopyranose in the backbone (Kacurakova et al., 2000). The spectral

region between 800 and 1200 cm− 1 is considered as “fingerprint” region

Fig. 3. The NMR spectra of POS4. (a) 1

H NMR spectrum; (b) COSY; (c) TOCSY; (d) HSQC.

J. Li et al.

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and varies among different polysaccharides (Hosseini et al., 2016). The

absorptions at around 886 cm− 1 and 762 cm− 1 were referred to α-type

glycosidic bonds, thus suggesting the presence of α-type glycosidic

linkage in POS4.

The structural characteristics of POS4 were further investigated

using the 1

H NMR spectra. As illustrated in Fig. 3a, the signal at 2.19

ppm was attributed to acetyl groups binding at 3-O-GalA. The peaks at

1.27 ppm and 1.18 ppm corresponded to the methyl groups of L￾rhamnose and were derived from the O-2 and O-2, 4 linked rhamnose,

respectively (Tamaki et al., 2008). The major peak at 3.61 ppm was

assigned to the methyl groups binding to carboxyl groups of GalA. Other

major signals in POS4 were derived from the five protons in the D￾Fig. 3. (continued).

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3.3.2. The intestinal damage and ROS level

Furthermore, to explore intestinal injury, 7-AAD was used to stain

the dissected guts of Drosophila. As shown in Fig. 6a, the number of dead

intestinal epithelial cells emitting red fluorescence was significantly

increased in the aging flies compared with the control flies according to

fluorescence intensity. Compared with the control group, the mean

fluorescence intensities of the aging flies in the experimental group

decreased by 53.43 % (p < 0.01) (Fig. 6b). Taking these findings into

consideration, POS4 could effectively protect intestinal integrity and

morphology of aging fruit flies.

With aging, the level of ROS increases dramatically and results in

oxidative stress. Excessive ROS damages cellular proteins, including

cytoskeletal proteins, and ultimately interrupts the gastrointestinal

barrier, thus increasing gut permeability and activating signaling path￾ways associated with the release of proinflammatory substances and

shortening lifespan (Qi et al., 2012; Xing et al., 2012). DHE can freely

enter cells through living cell membranes and is oxidized by intracel￾lular ROS to form ethyl oxide. Ethyl oxide can be incorporated into

chromosomal DNA to produce red fluorescence. According to the pro￾duction of red fluorescence in living cells, the ROS content in cells can be

determined. Therefore, we further estimated the effect of POS4 on the

ROS content in Drosophila. Compared with control group, the mean

fluorescence intensities of the flies fed POS4 decreased by 29.83 %

(Fig. 6c, d). Pectic polysaccharide is known to possess antioxidant ac￾tivity. In vitro, pectic polysaccharide could scavenge superoxide and

hydroxyl radicals and the content of GalA was positively correlated with

its antioxidant activity (Li, Qi, & Jasper, 2016). Moreover, pectic poly￾saccharide also ameliorated colon cancer by acting on oxidative stress￾and inflammation-activated signaling pathways in vivo (Tan et al.,

2018). In the present study, we have shown that galacturonic acid

enriched low molecular weight pectic polysaccharide could decrease

levels of intestinal ROS in flies.

Fig. 7. Effects of POS4 on the intestinal microbiota of aging male D. melanogaster. The effect of POS4 on the α-diversity, Shannon (a) and Simpson (b) index of midgut

microbiota. The effect of POS4 on the β-diversity of midgut microbiota, and PCoA was based on the weighted UniFrac distance (c) and NMDS Analysis (d); Bacterial

phylum abundance heatmap (n = 3) (e) and genus abundance (n = 3) (f); LEfSe result included the cladogram (g) and main dominant bacteria (h). The cladogram in

panel g shows the relative abundance of dominant bacteria in the Drosophila intestine that met a significant LDA threshold value of >2 in the POS4 intervention and

control groups. The bacterial community from the phylum to the genus level is provided from the center to the outside, respectively. Different colors represent

different groups, and the yellow node represents a group of microbes that do not play important roles in the different groups. A LEfSe analysis of the 16S sequence

was used to obtain a taxonomic cladogram and estimate the effective proportional abundance of each component. The lengths represent the effect size of the bacterial

lineages (panel h). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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3.3.3. The intestinal epithelial cells proliferation and differentiation

The homeostasis of intestinal barrier is based on a delicate regulation

of epithelial proliferation and differentiation. The excessive generation

of ROS with aging could result in the oxidative stress, contributing to the

excessive proliferation and differentiation of intestinal stem cells (ISCs),

thereby impairing gut homeostasis and affecting lifespan (Funk et al.,

2020). Therefore, the proliferation and differentiation of ISCs with POS4

intervention was determined. Notably, esgGFP+ cells in the aging flies

were assemble, expanded in size, and abnormal in morphology, and the

number and size of GFP+ cells were significantly enhanced (Fig. 6e).

Obviously, the amount and size of ISCs with POS4 supplement was

remarkably depressed by 41.78 % (P < 0.01) in contrast to control group

(Fig. 6f). The Drosophila adult midgut is maintained by ISCs that self￾renew and undergo mitosis to generate differentiated cell types of the

intestinal epithelium for gut replenishment. Therefore, PH3 antibodies

were applied to evaluate ISC division. As expected, in comparison with

control group, the amount of PH+ cells marking differentiated cells in

sample group with POS4 intervention was visibly decreased (Fig. 6g, h),

suggesting that POS4 can promote intestinal homeostasis by attenuating

aberrant proliferation and differentiation of ISC cells in the aging flies.

3.4. Effects of POS4 on the intestinal microflora of aging D. melanogaster

It has been suggested that aging-related intestinal barrier dysfunc￾tion is closely associated with disordered gut microbiota. As shown in

Fig. 7a, b, there were significant differences between Shannon and

Simpson indices of gut microbiota abundance in different fly groups.

Principal coordinate analysis (PCoA) indicating a similarity in micro￾biota composition showed an obvious clustering of gut microbiota

compositions between the two groups (Fig. 7c). As expected, NMDS

results (Fig. 7d) exhibited the similar tendency. Considering all results, it

was deduced that the composition of the intestinal microflora could be

altered by POS4 intervention in the aging D. melanogaster. Therefore, the

composition of gut microbiota in different groups is analyzed below at

phylum and genus levels.

As depicted in Fig. 7e, Proteobacteria, Firmicutes and Actinobacteria

are the predominant phylum. Compared with control group, POS4

intervention could decreased the abundances of Proteobacteria, Actino￾bacteria and Cyanobacteria, while increased the abundances of Firmicutes

and Bacteroidetes. At genus level, POS4 intervention could significantly

increase the proportion of Wolbachia from 40.07 % to 55.73 % (P >

0.05) and decrease the abundance of Commensalibacter, Pseudomonas,

Gluconobacter and Ralstonia (Fig. 7f). Furthermore, a linear discriminant

analysis (LDA) effect size (LEfSe) analysis was performed to reveal the

specific ba

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