Effect of enzyme-assisted extraction on the physicochemical properties and bioactive potential of lotus leaf polysaccharides

Journal Pre-proof Effect of enzyme-assisted extraction on the physicochemical properties and bioactive potential of lotus leaf polysaccharides

Young-Ran Song, Ah-Ram Han, Seul-Gi Park, Chang-Won Cho, Young-Kyoung Rhee, Hee-Do Hong PII:

S0141-8130(19)37650-0

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.02.252

Reference:

BIOMAC 14869

To appear in:

International Journal of Biological Macromolecules

Received date:

20 September 2019

Revised date:

2 February 2020

Accepted date:

22 February 2020

Please cite this article as: Y.-R. Song, A.-R. Han, S.-G. Park, et al., Effect of enzymeassisted extraction on the physicochemical properties and bioactive potential of lotus leaf polysaccharides, International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/j.ijbiomac.2020.02.252

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier.

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Effect of enzyme-assisted extraction on the physicochemical properties and bioactive potential of lotus leaf polysaccharides

Young-Ran Song, Ah-Ram Han, Seul-Gi Park, Chang-Won Cho, Young-Kyoung Rhee, and

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Hee-Do Hong*

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Korea Food Research Institute, Wanju-gun, Jeollabuk-do 55365, Republic of Korea

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*Correspondence: Hee-Do Hong

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Korea Food Research Institute, Wanju-gun, Jeollabuk-do 55365, Republic of Korea

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Tel +82-63-219-9285, Fax +82-63-219-9876, E-mail: [email protected]

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ABSTRACT Lotus leaf polysaccharides were extracted by enzyme-assisted extraction using α-amylase (LLEP-A), cellulose (LLEP-C), pectinase (LLEP-P) or protease (LLEP-PR). Their physicochemical properties and immunostimulatory activities were compared with those of hot-water extracted polysaccharides (LLWP). HPAEC-PDA and HPSEC-RI profiles indicated that variations in their molecular weight patterns and chemical compositions. Moreover, their

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effects on proliferation, phagocytic activity, and cytokine production in macrophages could

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be ordered as LLEP-P > LLEP-C > LLEP-A > LLWP > LLEP-PR, suggesting that LLEP-P by pectinase-assisted extraction was the most potent enhancer of macrophage activation.

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LLEP-P was further purified by gel filtration, and the main fraction (LLEP-P-І) was obtained

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to elucidate the structural and functional properties. LLEP-P-І (14.63 × 103 g/mol) mainly

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consisted of rhamnose, arabinose, galactose, and galacturonic acid at molar percentages of 15.5:15.8:20.1:32.8. FT-IR spectra indicated the predominant acidic and esterified form,

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suggesting the pectic-like structure. Above all, LLEP-P-І exerted greater stimulation effects

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on NO and cytokines production and the phagocytic activity in macrophages. Transcriptome analysis also demonstrated that LLEP-P and LLEP-P-І could upregulate macrophage immune response genes, including cytokines, chemokines, and interferon via TLR and JAK-STAT signaling. Thus, these results suggest that pectinase application is most suitable to obtain immunostimulatory polysaccharides from lotus leaves.

Keywords: Lotus leaf polysaccharide; Enzyme-assisted extraction; Immunostimulatory activity

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1. Introduction Immunomodulation refers to the process for maintaining immune homeostasis by boosting or suppressing the immune response [1]. Immunostimulation is an effective way to enhance the immune competency and disease resistance, while immunosuppression is often used to prevent tissue damage caused by an excessive response [2]. Especially in therapeutic approaches, immunostimulation induced by natural active substances is one of the most

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attractive topics for researchers [3]. In recent decades, natural polysaccharides have attracted

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attention as ideal immunostimulants due to their broad spectrum of immune activities as well

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as safety and biocompatible properties [4]. Many studies demonstrated that plant-derived polysaccharides can enhance the immune functions by triggering cellular/molecular events

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and strengthening innate and adaptive immune responses [3–6]. Various types of immune

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cells such as macrophages, dendritic cells, and lymphocytes can be activated by

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immunostimulatory polysaccharides [7]. In addition, the specific biological activities of polysaccharides are associated with their distinct structural features such as the

groups [5].

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monosaccharide and glycosidic-linkage composition, molecular weight, and functional

Lotus (Nelumbo nucifera Gaertn.), an aquatic herbal plant of the Nelumbonaceae family, is cultivated throughout Asia [8]. Its roots, seeds, and leaves are not only consumed as foods, but also used as traditional medicines [9]. Studies have demonstrated that lotus leaves possess anticancer [10], antidiabetic [8], anti‐ inflammatory [11], hepatoprotective, and antioxidant [9] activities. Thus, lotus leaf preparations are increasingly used in health-promoting foods and supplement formulations [9]. Recent research demonstrated that polysaccharides extracted from different parts of the lotus are associated with several bioactivities [8,12–14]. In our earlier study, water-soluble polysaccharide (LLWP) having immunostimulatory 3

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potential was obtained from lotus leaves by hot-water extraction, and the two most abundant polysaccharide fractions were further purified [15]. Extraction process is the first and important step in the study of biological polysaccharides in natural resources. Typical polysaccharide extraction methods include maceration, hotwater extraction (HWE), chemical extraction, and acid hydrolysis. However, these methods usually require high temperature, long processing time, and/or large solvent volumes, which

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can lead to co-extraction of many non-active components and adversely affect the active

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ingredients [16]. Other techniques, such as microwave, ultrasonication, and enzymatic

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digestion, have been tested recently for developing faster and/or more efficient polysaccharide extraction protocols [17]. Among those, the enzyme-assisted extraction (EAE)

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has been frequently used because it is environmentally compatible and more selective for

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extracting target compounds from source materials [18]. Enzymes such as α-amylase,

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cellulase, pectinase, and protease are frequently used to extract polysaccharide from plant sources [17,19]. Specifically, the enzymatic assistance can improve the extraction efficiency

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and biological activities of the polysaccharides [19,20]. The effectiveness of EAE depends greatly on the source material [16]. Previous studies reported that EAE method led to higher yields of polysaccharides from plants such as the fruit of Cornus officinalis and naked pumpkin seeds [18,21]. In addition, antioxidant activity of polysaccharides from Astragalus membranaceus and immunomodulatory activities of polysaccharides from Dendrobium chrysotoxum and Panax ginseng were contributed by EAE technology [16,22,23]. Moreover, EAE can be applied to obtain different polysaccharides from the same material [18]. However, to our knowledge, there are no published studies on using EAE to recover polysaccharides from lotus.

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In this study, the potential applicability of EAE using different commercial enzymes was explored for efficient extraction of lotus leaf polysaccharides by comparing the yields and evaluating the structural and immunostimulatory properties. Moreover, their physicochemical and biological properties were compared with those obtained by a traditional HWE method. These results might provide a better exploration for extraction of biological lotus leaf

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polysaccharides.

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2. Materials and methods

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2.1. Materials

Lotus (Nelumbo nucifera Gaertn.) leaves were purchased from Kyungdong market, Seoul,

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Korea, and dried and ground with a grinding mill. The α-amylase (EC 3.2.1.1) from

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Aspergillus oryzae (Diazyme®; optimal temperature, 50 C ; optimal pH, 5.0), cellulase (EC 3.2.1.4) from Aspergillus oryzae (Cellulase KN; optimal temperature, 40–50 C ; optimal pH

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4.0–6.0; activities: cellulase and hemicellulase), pectinase (EC3.2.1.15) from Aspergillus niger (Plantase MAX®; optimal temperature, 10–55 C; optimal pH, 3.0–5.0; activities: polygalacturonase and pectinesterase), and protease (EC 3.4.21.62 and EC 3.4.24.28) from Bacillus amyloliquefaciens (Protex 7L; optimal temperature, 50–70 C; optimal pH, 6.5–7.5; activities: endopeptidase) were purchased from Vision Corporation (Seongnam, Gyonggi, Korea). All solvents and reagents for the chemical analysis were of analytical grade.

2.2. Extraction of polysaccharides from lotus leaves

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The EAE-derived polysaccharides (LLEPs) from lotus leaves were prepared using four different commercial enzymes. Dried lotus leaves (100 g) were crushed and dispersed in 2 L of distilled water (solid/liquid ratio, 1:20 w/v). The pH of the suspension was adjusted to either pH 4.5–5.0 for amylase, cellulase, and pectinase, or pH 7.0 for protease. The extraction was conducted at 50 C for 48 h after adding each enzyme (1%, v/w of raw material). Then, the suspensions were rapidly heated at 95 C for 20 min for enzyme inactivation.

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Supernatants were collected by centrifugation at 6000 ×g and filtered through Whatman No.

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2. Afterwards, cold ethanol was added to the aqueous extract to form a final ethanol

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concentration of 75% (v/v), and kept at -20 C overnight. The precipitate was obtained, dissolved in water, and dialyzed using a Spectra/Por® 6000–8000 Da cut-off membrane

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(Spectrum Laboratories Inc., Rancho Dominguez, CA, USA). Then, the retentate (high-

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molecular-weight fraction) was lyophilized to give LLEPs: specifically, LLEP-A by α-

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amylase, LLEP-C by cellulase, LLEP-P by pectinase, and LLEP-PR by protease. The HWE-derived polysaccharide (LLWP) from lotus leaves was prepared as reported

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previously [15]. Briefly, lotus leaves were mixed with 20 volumes of distilled water and extracted twice under reflux conditions at 100 C for 4 h each. The crude polysaccharides was separated from the aqueous supernatants by ethanol precipitation. After dialysis using a Spectra/Por® 6000–8000 Da cut-off membrane, the high-molecular-weight fraction was lyophilized to obtain LLWP.

2.3. Evaluation of physicochemical properties of four LLEPs and LLWP 2.3.1. Analytical methods

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The contents of neutral sugar and uronic acid were determined using the phenol-sulfuric acid method [24] and sulfuric acid-carbazole method [25], respectively. Protein concentration was measured using the Bradford method [26]. The modified thiobarbituric acid-positive method [27] was used to determine the content of 2-keto-3-deoxy-D-mannooctanoic acid

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(KDO)-like materials in polysaccharide samples.

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2.3.2. Monosaccharide composition analysis

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Monosaccharide composition was determined by high-performance anion-exchange chromatography (HPAEC)-PAD using an ICS-5000 Dionex chromatography system (Dionex,

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Sunnyvale, CA, USA) after acidic hydrolysis of polysaccharides in 2 M trifluoroacetic acid at

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100 C for 4 h. Run was performed with a PA1-precolumn (4 × 50 mm, Dionex, USA) and a CarboPac™ PA-1 analytical column (4 × 250 mm, Dionex, USA) at 30 °C. Neutral sugars

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were separated using 18 mM NaOH, and uronic sugars were eluted with 100 mM NaOAc in

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100 mM NaOH at a constant flow rate of 1.0 mL/min. Monosaccharides were identified by their elution times relative to mixes of sugar standards, including arabinose, fucose, galactose, glucose, mannose, rhamnose, xylose, glucuronic acid, and galacturonic acid.

2.3.3. Molecular weight determination To determine the molecular weight patterns of polysaccharide samples, high-performance size-exclusion chromatography (HPSEC) was conducted using an HPLC system (JASCO PU-2089 Plus, Jasco, Tokyo, Japan) equipped with a RI detector (JASCO RI-2031 Plus, Jasco, Tokyo, Japan) and two tandemly linked columns, Asahipak GS-520 and Asahipak GS-320 7

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(7.6 mm × 30 cm each, Showa Denko Co., Tokyo, Japan). The sample (10 mg/mL) was eluted with 50 mm ammonium formate (pH 5.5 with formic acid) at a flow rate of 0.4 mL/min at 40 °C. Standard pullulan series (P-336, P-113, P-48.8, P-21.7, P-10, P-6, P-1.32, and P-0.342) were used for calibration.

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2.4. Evaluation of immunostimulatory effects of four LLEPs and LLWP

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2.4.1. Cell culture

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RAW 264.7 murine macrophage cells were purchased from Korean Cell Line Bank (Seoul,

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Korea) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%

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2.4.2. Cell viability assay

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with 5% CO2 atmosphere.

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fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified incubator at 37 °C

The effects of lotus leaf polysaccharides on RAW264.7 cell viability were evaluated using a conventional Cell Counting Kit-8 (CCK‐ 8; Dojindo, Tokyo, Japan). Briefly, the macrophage cells (2 × 105 cells/mL) were pre-cultured in 48-well plates for 24 h, and then treated with different concentrations of polysaccharide samples (3, 10, and 30 μg/mL) or LPS (1 μg/mL) for 24 h. Then, 20 μL/well of CCK‐ 8 solution was added to the cultured cells, and the cytotoxic effects were determined, per the manufacturer’s instructions.

2.4.3. In vitro macrophages phagocytosis assay 8

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The efficacy of lotus leaf polysaccharides on the phagocytic activity of macrophages was measured using the neutral red uptake assay [28]. RAW 264.7 cells were stimulated by polysaccharide samples or LPS, as mentioned above. Then, their supernatants were discarded. Aliquots of 200 μL 0.075% neutral red solution were added to each well, and incubation continued for 30 min. Then, the supernatants were discarded, and the cells were washed with PBS three times. After 200 μL of cell lysis solution (1 M glacial acetic acid:ethanol = 1:1, v/v)

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was added into each well, the plate was stored for 1 h to extract the absorbed neutral red. The

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absorbance was measured at 540 nm, and the phagocytic index was calculated using the

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2.4.4. NO and cytokine secretion assay

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following equation: Abs sample/Abs blank control.

After RAW 264.7 cells were treated with polysaccharide samples for 24 h as described

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above, each culture supernatant was collected and subjected to NO and cytokine analysis. To

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determine total NO level, equal volumes of Griess reagent (Sigma) was added to collected supernatant, and the reaction continued in the dark at RT for 15 min. The contents of nitrite were determined by measuring the absorbance at 540 nm based on a standard curve plotted with sodium nitrite. In addition, cytokine TNF-α, IL-1β, and IL-6 levels were assessed using corresponding immunoenzymatic assay (ELISA) kits (BD Biosciences, Pharmingen, CA, USA) according to the manufacturer’s instructions. The optical density was determined at 450 nm.

2.5. Purification and characterization of LLEP-P

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2.5.1. Purification of LLEP-P LLEP-P was further purified by gel permeation chromatography using a Sephadex G-100 column (2.5 cm i.d. × 90 cm). Ammonium formate (pH 5.5 with formic acid) was used at a flow rate of 0.6 mL/min. The resulting eluates (6 mL/tube) were collected by a fraction collector (Bio-rad, Hercules, CA, USA) and then monitored for the carbohydrate content by the phenol-sulfuric acid method. As the main fraction, LLEP-P-І was pooled, concentrated,

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dialyzed using a Spectra/Por 6000–8000 Da cut-off membrane against distilled water and

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then lyophilized.

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2.5.2. Physicochemical analysis

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The contents of neutral sugar, uronic acid, protein, and KDO-like materials of the purified

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LLEP-P-І were determined using the analytical methods as described above (see Section 2.3.1), and the monosaccharide composition was determined by HPAEC-PAD analysis (see

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Section 2.3.2). In addition, homogeneity and molecular weight of LLEP-P-І were determined by high-performance gel permeation chromatography (HPGPC)-RI analysis using a Tosoh EcoSEC HLC-8320 GPC system (Tosoh Bioscience, Tokyo, Japan). The column set consisted of TSKgel guard PWXL, two TSKgel GMPWXL, and TSKgel G2500PWXL (7.8 × 300 mm) was used and maintained at 40 °C. The polysaccharide sample (3 mg/mL) was eluted with a 0.1 M NaNO3 at a flow rate of 1.0 mL/min. Data acquisition was performed using EcoSEC software with a standard curve prepared from pullulan standards to obtain molecular weight. UV-visible spectroscopic analysis of LLEP-P and LLEP-P-І were conducted using UV‐ Vis spectroscopy (Beckman Coulter DU 800; Beckman Coulter, Fullerton, CA, USA) in the wavelength range of 200-800 nm. FT-IR analysis was recorded with a Fourier Transform 10

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Infrared Spectrometer (FTIR-4600; Jasco, Tokyo, Japan) at the frequency range of 4000– 500 cm−1 using the KBr-disk method.

2.5.3. Analysis of immunostimulatory properties The effects of polysaccharide samples (LLWP, LLEP-P, LLEP-P-І, or LPS) on cell viability,

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phagocytosis activity, and production of NO and cytokines in RAW 264.7 macrophage cells

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were evaluated as described above (Section 2.4), and compared.

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Furthermore, the effects of lotus leaf polysaccharides on gene expression in the

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macrophage cells were determined by transcriptome sequencing (RNA-seq analysis). Firstly, total RNA was isolated from RAW 264.7 cells stimulated with 30 μg/mL of polysaccharide

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samples (LLWP, LLEP-P, or LLEP-P-І) or 1 μg/mL of LPS using NucleoSpin RNA

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(Macherey-Nagel, Duren, Germany). RNA quality was evaluated with Agilent 2100 bioanalyzer using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The

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Netherlands), and RNA quantification was performed using ND-2000 Spectrophotometer (Thermo Inc., DE, USA). For control and test RNAs, sequencing libraries were generated using QuantSeq 3’ mRNA-Seq Library Prep Kit for Illumina (Lexogen, Inc., Austria) using 500 ng of total RNA per sample [29]. Briefly, an oligo-dT primer containing an Illuminacompatible sequence at its 5’ end was hybridized to the RNA, and reverse transcription was performed. After degradation of the RNA template, second strand synthesis was initiated by a random primer containing an Illumina-compatible linker sequence at its 5’ end. The doublestranded library was purified using magnetic beads to remove reaction components. The library was amplified to add the adapter sequences required for cluster generation. The finished library was purified from PCR components. High-throughput sequencing was 11

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performed as single-end 75 sequencing using NextSeq 500 (Illumina, Inc., USA). To read the mRNA-seq, mapping was performed using TopHat to obtain sorted files [30]. Differentially expressed genes (DEG) were determined based on counts from unique and multiple alignments using coverage in Bedtools [31]. The read count (RC) data were processed based on a quantile normalization method using EdgeR within R (R development Core Team, 2016) and Bioconductor [32]. Up- or down-regulated genes were identified using ExDEGA v.1.6.3

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(E-biogen Inc., Seoul, Korea). Gene classification was based on searches of DAVID

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(http://david.abcc.ncifcrf.gov/). The Gene Ontology (GO) from the DAVID analysis was used to show the correlation in Quick GO (https://www.ebi.ac.uk/QuickGO). The clustering heat

2.6. Statistical analysis

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maps of DEGs were generated by MeV v.4.9 software program.

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All samples were processed in triplicate, and values are expressed as the mean ± SD. Using

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statistical software SPSS (Version 20.0, Chicago, IL, USA), data were subjected to unpaired t-tests or one-way analysis of variance (ANOVA), followed by Duncan’s multiple range tests. A p < 0.05 was considered statistically significant.

3. Results and discussion 3.1. Preparation of LLEPs In this study, four different LLEP samples (LLEP-A, LLEP-C, LLEP-P, and LLEP-PR) were prepared by EAE using α-amylase, cellulase, pectinase, and protease, respectively, whereas LLWP was obtained by HWE and used for comparison. Here, the yield of LLWP 12

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was 1.18% (Table 1). In addition, LLEP-A, LLEP-C, LLEP-P, and LLEP-PR had yields of 0.97%, 1.17%, 1.11%, and 1.93%, respectively. All enzymatic extractions, except that performed with protease, did not significantly affect the polysaccharide yields. Meanwhile, the yield of LLEP-PR was apparently higher than LLWP. The results suggested that enzymatic extraction efficiency differed among the four enzymes in which only protease can

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increase the extraction yields of lotus leaf polysaccharides, compared with HWE.

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3.2. Comparison of chemical composition of LLWP and LLEPs

The chemical and monosaccharide compositions of LLEPs were analyzed and compared

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with LLWP (Table 1). In all extracts, the total sugar levels based on neutral sugar and uronic

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acid contents were above 96%. Compared with LLWP, four LLEPs contained significantly higher neutral sugar and lower uronic acid contents. Particularly, LLEP-PR has the highest

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neutral sugar and the lowest uronic acid levels. In addition, the protein content was the

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highest in LLEP-PR. Meanwhile, LLEP-P contained more KDO-like materials than other extracts. Typically, polysaccharides are constituted by different monosaccharides, which are the basic units that form unique structures and properties [33]. KDO is also well known for a unique component of rhamnogalacturonan-II (RG-II) among many plant polysaccharides [5]. Here, the monosaccharide compositions of five polysaccharides were determined by HPAEC. Obviously, their sugar profiles were different (Table 1). The lotus leaf polysaccharides were mostly consisted of rhamnose, arabinose, galactose, glucose, and galacturonic acid. However, their molar ratios were significantly different in the extracts; 5.95:15.05:19.51:12.31:36.17 (LLWP), 8.52:22.63:24.94:2.93:30.63 (LLEP-A), 8.21:22.89:26.18:4.02:29.66 (LLEP-C), 11.90:21.21:29.77:3.50:21.80 (LLEP-P), and 4.18:16.63:22.86:26.51:13.88 (LLEP-PR), 13

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respectively. Compared with LLWP, the levels of rhamnose, arabinose, and galactose were significantly higher in LLEP-A, LLEP-C, and LLEP-P. Specially, LLEP-P had the highest levels. Meanwhile, LLEP-PR showed lower levels of rhamnose, arabinose, and galacturonic acid with remarkably higher glucose and glucuronic acid levels, indicating a distinct change among all extracts. Plant cell walls are complex and dynamic structures formed by a heterogeneous mixture of cellulose, hemicelluloses, and pectins, along with ~10% structural

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glycoproteins such as extensins and arabinogalactan-proteins [34]. Of these, pectic

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polysaccharides contain a high proportion of galacturonic acid, which is mainly substituted by rhamnose residues with side chains such as rhamnose, arabinose and galactose [5]. Thus,

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we confirmed that pectic polysaccharides were present as the main polysaccharides of lotus

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leaves, which is consistent with previous studies [15]. Moreover, monosaccharide

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compositions of the polysaccharides could be affected by different enzymes and extraction methods. The effect by protease was obviously different from the other three enzymes. One

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possible explanation would be that LLEP-PR contains relatively higher proportions of

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arabinose, galactose, glucose, and glucuronic acid as well as protein, which might indicate the presence of structural glycoproteins such as arabinogalactan-proteins in LLEP-PR [35]. In addition, our results suggest the more abundant RG-II polysaccharide in LLEP-P, considering that LLEP-P had higher contents of KDO-like materials, rhamnose, arabinose, and galactose.

3.3. Comparison of molecular weight profiles of LLWP and LLEPs Furthermore, molecular weight profiles of the extracts were evaluated by HPSEC, and their average molecular sizes were determined relative to pullulan standards. As shown in Fig. 1, all extracts possessed several peaks indicating the highly polydisperse feature. However, their patterns were apparently different according to extraction methods. LLWP showed five 14

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distinct groups, with average molecular weights of 70.6, 15.5, 1.88, 1.32, and 0.73 × 103 Da. While, LLEP-A and LLEP-C had more carbohydrate fractions (399, 233, 79.1, 16.5, 3.59, 2.52, 1.41, and 0.55 × 103 Da in LLEP-A; 150.8, 81.6, 15.8, 4.05, 2.43, 1.63, and 0.61 × 103 Da in LLEP-C), possibly due to the enzymatic cleavage to release more polymer segments [20]. In addition, LLEP-P showed a large major peak with the average molecular weight value of 14.9 × 103 Da, and two small peaks at 76.8 and 1.11 × 103 Da. LLEP-PR exhibited

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two major peaks with average molecular weights of 89.9 and 48.4 × 103 Da, and smaller

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peaks with molecular weights of < 13.9 × 103 Da. Typically, pectic polysaccharides can have a wide molecular weight ranges of ~1600 × 103 Da, and their molecular weight distribution,

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structure, and size can affect the behavior of the polysaccharides [5]. In this study, lotus leaf

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polysaccharides had multiple molecular weight values, ranging between 399 and 0.61 × 103

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Da. Furthermore, we observed that molecular weight distributions of polysaccharides were markedly affected by different extraction treatments; specifically, α-amylase and cellulase

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digestions were attributed to obtain more complex polysaccharides from lotus leaf, whereas

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pectinase and protease caused simpler profiles.

3.4. Comparison of immunostimulatory activities of LLWP and LLEPs 3.4.1. Effects on macrophage viability and phagocytic activity As key immune effector cells, macrophages are essential for cell-mediated immunity which exert a variety of functions, including phagocytosis, biochemical destruction of the targeted organisms, and chemotaxis [36]. After activation, macrophages produce various molecules, including NO and cytokines, which enhance and regulate immune responses [37]. Therefore, macrophages represent an ideal cell model to assess the immunological activity of 15

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polysaccharides. As shown in Fig. 2A, none of the extracts had any cytotoxic effect on RAW 264.7 macrophages, and moreover they significantly enhanced cell proliferation within the tested concentration range (3, 10, and 30 μg/mL). Meanwhile, LLEP-PR had the lowest stimulatory effect among the extracts. Compared with untreated cells, lotus leaf polysaccharides enhanced the phagocytic activities, except for LLEP-PR showing no effect (Fig. 2B). Moreover, LLEP-A, LLEP-C,

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and LLEP-P had stronger effects than LLWP. Of these, LLEP-P exhibited the highest

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phagocytic activity at tested concentrations. Phagocytosis of particles and pathogens is a key

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function of macrophages in innate host defense, which mediates the adaptive response. Activated macrophages can destroy pathogens directly via phagocytosis and indirectly by

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secreting pro-inflammatory factors [38]. Thus, the results indicated that lotus leaf

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polysaccharides extracted with amylase, cellulase, and pectinase were better in promoting the

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phagocytic activity than LLWP. In particular, LLEP-P was the most potent enhancer of

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macrophage activation to activate, trigger, and increase phagocytosis.

3.4.2. Effects on the production of NO and cytokine in macrophages Activated macrophages can also produce multiple mediator molecules, such as NO, IL-1β, IL-6, IL-10, and TNF-α, which are involved in host defense, inflammation, and immunological diseases [28]. In this study, LLWP, LLEP-A, LLEP-C, and LLEP-P stimulated NO release from macrophages (Fig. 2C), and the highest NO level was induced by LLEP-P treatment (p < 0.05). In contrast, LLEP-PR did not affect the NO secretion. Similar results were observed in the cytokine secretion (Fig. 2D-F). Compared with untreated cells, the polysaccharides significantly stimulated TNF-α, IL-1β, and IL-6 secretion in a dose16

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dependent manner, except LLEP-PR. Of note, LLEP-P was the most effective. Taken together, the effect of the extracts on enhancing cell proliferation, phagocytic activity, and production of NO and cytokines in macrophages could be summarized as follows: LLEP-P > LLEP-C > LLEP-A > LLWP > LLEP-PR. This suggested that enzymatic digestion using amylase, cellulase, and pectinase can be a useful method to extract immunostimulatory polysaccharides from lotus leaf. Overall, a greater positive effect on the macrophage

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activation was observed in LLEP-P by pectinase-assisted extraction. In contrast, protease

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assistance rather given the adverse effect in these immunostimulating effect of lotus leaf polysaccharide. It is well established that the immunostimulatory activity of specific

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polysaccharides are closely related to the chemical and structural features, including

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monosaccharide and glycosidic composition, molecular weight, and side-chain distribution

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[5]. Here, the low immunostimulatory activity of LLEP-PR might be related to the notable difference in the chemical and structural features, including its distinctive chemical

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compositions and molecular weight profile. In addition, our results suggest that the

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immunostimulatory properties of lotus leaf polysaccharides might be attributed to pectic polysaccharides rather than glycoproteins.

3.5. Purification and characterization of LLEP-P 3.5.1. Physicochemical characteristics LLEP-P, which has the strongest immunostimulatory effect, was further purified by gel filtration on a Sephadex G-100 column (Fig. 3A). As the main fraction, LLEP-P-І was obtained with the final recovery rate of 48.3%, and the physicochemical and functional properties were elucidated. The molecular weight of the purified LLEP-P-І was evaluated by 17

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HPGPC-RI analysis, and the chromatogram for LLEP-P-І showed a single and symmetrical peak (Fig. 3B and Fig. S1). The number-average molecular weight (Mn), the weight-average molecular weight (Mw), and Z-average molecular weight (Mz) of LLEP-P-І were calculated to be 11.55, 14.63, and 17.40 × 103 g/mol, respectively. It was consistent with earlier results on the molecular weight patterns of LLEP-P (Fig. 1E). LLEP-P-І has relatively lower molecular weight compared to other immunostimulatory pectin polysaccharides and

of

arabinogalactan proteins with the molecular weight ranges 4–8400 kDa [5]. The

ro

polydispersity indicates the width of the molecular weight distribution and low polydispersity values reflect narrow molecular weight distributions [39]. Here, the polydispersity coefficient

-p

of LLEP-P-І was 1.27 (Mw/Mn) or 1.19 (Mz/Mw), indicating a homogeneous polysaccharide.

re

In addition, the chemical composition analysis showed that LLEP-P-І was composed of

lP

neutral (52.71%) and acidic (43.50%) carbohydrates with 3.79% of KDO-like materials. Particularly, in comparison to crude LLEP-P, the proportion of KDO-like materials in LLEP-

na

P-І was the higher, suggesting that RG-II-rich structure of LLEP-P was derived from LLEP-

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P-І [40]. The monosaccharide profile revealed that LLEP-P-І mainly consisted of rhamnose (17.58 mol%), arabinose (25.90 mol%), galactose (17.27 mol%), and galacturonic acid (27.82 mol%), with lower amounts of fucose (1.98 mol%), glucose (1.73 mol%), mannose (4.48 mol%), xylose (1.35 mol%), and glucuronic acid (1.89 mol%), indicating higher fucose, rhamnose, arabinose, and galacturonic acid levels, compared with LLEP-P (Fig. 3C and 3D). In addition, no absorption at 260 nm and 280 nm indicated the absence of protein and nucleic acid in LLEP-P-І, unlike crude LLEP-P (Fig. 3E). FT-IR analysis was performed, and the overall spectrum of LLEP-P and LLEP-P-І were similar, showing typical absorption bands of polysaccharides (Fig. 3F). In fact, a large absorption peak between 3600–3200 cm-1 was indicative of the hydroxyl group (O–H). The 18

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weak band at 2934 cm-1 was allotted to C–H stretching vibration of CH, CH2, and CH3. The relatively strong absorption near 1600 cm-1 could be assigned to the deformation vibrations of water (H2O) overlapping with the antisymmetric stretching vibration of carboxylate anion (COO-) [41]. The peak at 1733 cm-1 was ascribed to the stretching of the C=O bond of the ester carbonyl group, and the additional bands around 1416, 1373, and 1341 cm-1 suggested the presence of free or esterified carboxyl groups and C–H bending vibration of a pyranoic

of

ring [42]. The results indicated the presence of uronic acid residues in the polysaccharides, in

ro

agreement with the chemical analysis. The absorption near 1244 cm-1 suggested the presence of S=O bonds [43]. In addition, the strong characteristic bands around 1200–950 cm-1 were

-p

derived from the overlapping vibrations of ring and side group vibrations of C–C, C–O, and

re

C–OH with the C–O–C glycoside ring bond, indicating the existence of pyranose sugars [43].

lP

Moreover, LLEP-P-І showed absorption peaks around 917 cm-1 and 812 cm-1, which might be due to the presence of β-linkages and α-linkages of pyranoses in the polysaccharides [44].

na

Overall, the FT-IR spectra of LLEP-P and LLEP-P-І showed the presence of predominantly

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acidic and esterified forms, which are characteristic for pectic polysaccharides [42].

3.5.2. Distinguishing the immunostimulatory properties To confirm and explain the enhanced immunostimulatory activity of LLEP-P in RAW 264.7 macrophages, the effects of purified LLEP-P-І on cell viability, phagocytosis activity, and production of NO and cytokines were evaluated and compared with crude LLEP-P and LLWP. As shown in Fig. 4A, LLEP-P-І was not cytotoxic to the cells for all the doses tested (1-100 μg/mL). LLEP-P-І was better in promoting the phagocytic activity of the macrophage than crude LLEP-P as well as LLWP (Fig. 4B). Moreover, LLEP-P-І exerted greater stimulatory effects on NO, TNF-α, IL-1β, and IL-6 secretion (Fig. 4C-F). Thus, these results 19

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indicated that the higher immunostimulatory property of LLEP-P compared with LLWP was attributed to the main fraction, LLEP-P-І. In particular, the levels of NO, TNF-α, and IL-6 by LLEP-P-І (above 10 μg/mL) were comparable to those of LPS used as positive control (1.0 μg/mL). Further experiments on the immunostimulatory properties of LLEP-P and LLEP-P-І (at 30 μg/mL) were conducted using RNA sequencing analysis to evaluate their transcriptional

of

regulation in the macrophages [29], compared with those in LLWP. Gene expression changes

ro

in macrophages treated with LLWP, LLEP-P, LLEP-P-І or LPS were determined by

-p

comparison with the untreated control, and then their effects on the cellular responses in the macrophages were compared. We specifically focused on the expression patterns of a total of

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804 genes involved in immune response. Among those, 171 (21.27%), 205 (25.50%), 256

lP

(31.84%), and 302 (37.56%) genes were regulated by treatments of LLWP, LLEP-P, LLEP-P-

na

І, and LPS, respectively, which were up- or downregulated with ≥ 2.0-fold change in treated RAW 264.7 cells (Fig. 5A). As shown in Fig. 5B, a cluster heat map for the expression levels

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of the 344 differentially expressed genes also displayed that the transcriptional changes induced by LLEP-P and LLEP-P-І were markedly higher than that by LLWP. Moreover, the results on similarity among samples showed that the LLEP-P-І-induced transcriptome response was similar to the LPS-induced transcriptome. Of genes induced by the samples, representative expression profiles are presented in Fig. 5C. Certainly, lotus leaf polysaccharides induced the expression of genes encoding various cytokines and chemokines, including IL-1, IL-4, IL-6, TNF, CCL1, CCL2, CCL7, CXCL2, and G-CSF (CSF3), as well as various interferon response genes. However, the up-regulating effects were markedly higher by LLEP-P and LLEP-P-І, while LLWP showed a relatively low induction. Thus, the gene expression-inducing effects were greatly enhanced by pectinase-assisted extraction. 20

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Interestingly, genes involved in JAK-STAT and TLR/NF-κB signaling pathways were also up-regulated by lotus leaf polysaccharides. In activated macrophages, the triggered cellular signaling pathways cause changes in the expression of various secondary mediators such as cytokines and chemokines, and these mediators are critical for initiating innate immunity, orchestrating adaptive immune mechanisms, and ultimately constraining immune responses [36,45,46]. In this study, lotus leaf polysaccharides could induce the expression of multiple

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genes, including various cytokines, chemokines, and interferon, by promoting JAK-STAT and

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TLR-mediated signaling pathways. Hence, we suggest that lotus leaf polysaccharides can stimulate innate immunity by inducing macrophage activation via regulating microRNA

-p

expression, and, moreover, these effects can be enhanced by applying an appropriate

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4. Conclusion

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extraction technique.

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In this study, an enzymatic extraction technique for lotus leaf polysaccharides was developed to improve their yields and biological activities. Extraction yields did not vary between EAE and HWE. However, their physicochemical and immunostimulatory properties differed significantly. Lotus leaf polysaccharides were mainly composed of rhamnose, arabinose, galactose, and galacturonic acid with different ratios according to the extraction methods. Interestingly, the different extractions resulted in distinct molecular weight distributions, in which LLEP-P showed a simpler pattern with the main fraction of approximately 15 kDa. Moreover, LLEP-P exerted the greatest stimulatory effect on RAW 264.7 macrophages in terms of NO and cytokine productions, phagocytic activity, and cell proliferation. Thus, we assumed that the enhanced immunostimulatory activity of LLEP-P might be derived from its main fraction. Accordingly, the main fraction (LLEP-P-І) was 21

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purified from LLEP-P, and the structural and biological characteristics were further confirmed. The chemical composition and FT-IR analysis indicated that LLEP-P-І consisted mainly of galactose, arabinose, rhamnose, and galacturonic acid, and had a predominant acidic and ester form, suggesting its pectic structure. Moreover, the high level of KDO-like materials in LLEP-P-І suggested that it is the RG-II-rich polysaccharide. Besides, the excellent immunostimulatory potential of LLEP-P-І was demonstrated though stimulatory

of

activity assays on macrophages, including transcriptome analysis. Taken together, our results

ro

suggest that enzymes could be employed to extract lotus leaf polysaccharides with desirable properties. Specifically, lotus leaf polysaccharides having the major structural characteristics

-p

importing immunostimulating properties can be selectively obtained through pectinase

re

assisted extraction. However, the link between structural features and immunological

lP

mechanism of LLEP-P-І are still unclear, and thus further study on the structure-activity

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Acknowledgements

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relationship is going on in our laboratory.

This research study was supported by the Main Research Program (E0164700) of the Korea Food Research Institute (KFRI) funded by the Ministry of Science and ICT.

Declarations of interest None.

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Tables Table 1. Chemical properties of polysaccharides from lotus leaves extracted using different methods. Sample

LLWP

LLEP-A

LLEP-C

LLEP-P

LLEP-PR

Yield (%)

1.18±0.30b 0.97±0.17b 1.17±0.16b 1.11±0.14b 1.93±0.15a

54.67c

65.36b

66.92b

68.66b

80.47a

Uronic acid

43.82a

32.00b

31.00b

28.70b

Protein

0.90d

2.04b

of

15.60c

1.32cd

1.52bc

3.31a

KDO3)-like material

0.61b

0.60b

0.76b

1.12a

0.62b

Fucose

0.77b

0.79b

0.77b

1.25a

0.36c

Rhamnose

5.95c

8.52b

8.21b

11.90a

4.18d

Arabinose

15.05b

22.63a

22.89a

21.21a

16.63b

Galactose

19.51d

24.94b

26.18b

29.77a

22.86c

Glucose

12.31b

2.93c

4.02c

3.50c

26.51a

3.85b

4.31a

2.82c

4.43a

1.80d

4.08a

2.55c

3.14b

2.08d

2.68c

Galacturonic acid

36.17a

30.63b

29.66b

21.80c

13.88d

Glucoronic acid

2.31c

2.71c

2.31c

4.06b

11.11a

1)

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Mannose Xylose

-p

re

lP

Component sugar (mol%) 4)

ro

Neutral sugar

na

Chemical composition (%) 2)

Values are expressed as the means ± SD (n=3). Different lowercase superscripted letters (a-

d) in rows indicate significant differences among the five different polysaccharides according to the extraction methods (p < 0.05). 2)

Percentage (%) in dried extract.

3)

KDO, 2-keto-3-deoxy-D-mannooctanoic acid.

4)

Mol% was calculated from the detected total neutral sugar. 30

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Figure legends Fig. 1. HPSEC profiles on molecular weight patterns of lotus leaf polysaccharides obtained by different extraction methods. (A) Pullulan standards, (B) LLWP obtained by hot-water extraction, and LLEP-A (C), LLEP-C (D), LLEP-P (E), and LLEP-PR (F) obtained by

of

enzyme-assisted extraction.

Fig. 2. Effects of lotus leaf polysaccharides obtained by different extraction methods on cell

ro

viability (A), phagocytic activity (B), and secretion levels of NO (C), TNF-α (D), IL-1β (E),

**

p < 0.01, and

***

p < 0.001. The lowercase letters (a–e) indicate

re

identified; *p < 0.05,

-p

and IL-6 (F) in macrophages. Significant differences compared to the control cells (NC) are

na

concentration.

lP

significant differences (p < 0.05) among the five different polysaccharide samples at the same

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Fig. 3. Elution profile (A) of LLEP-P on a Sephadex G-100 column, and HPGPC-RI chromatogram (B) for molecular weight determination of purified LLEP-P-І. HPAEC-PAD chromatograms on monosaccharide compositions of LLEP-P (C) and LLEP-P-І (D). UV (E) and FT-IR (F) spectrum of LLEP-P and LLEP-P-І.

Fig. 4. Effects of LLWP, LLEP, and LLEP-P-І on cell viability (A), phagocytic activity (B), and secretion levels of NO (C), TNF-α (D), IL-1β (E), and IL-6 (F) in macrophages. Significant differences compared to the control cells (NC) are identified; *p < 0.05, **p < 0.01, and ***p < 0.001. The lowercase letters (a–c) indicate significant differences (p < 0.05) among 31

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the three different polysaccharide samples at the same concentration.

Fig. 5. Effect of LLWP, LLEP, and LLEP-P-І on expression profile of genes related to immune response in macrophage cells followed by RNA-seq. (A) The counts of genes changed at least 2-fold among total 804 genes related to immune function in macrophages

of

treated with each polysaccharide sample. (B) Heat map of overlapping differentially expressed 344 genes from RNA-seq expression data set from polysaccharides-treated

ro

macrophages compared with the data set from untreated control cells among total 804 genes

-p

related to immune function. The mRNA differential expression levels in LPS-, LLWP-,

re

LLEP-P-, and LLEP-P-І-treated cells compared with untreated control cells, which changed

lP

for more than 2-fold up- and downregulation with p-value of less than or equal to 0.05. The red, white, and blue colors indicate up-expression, equal to, and down-expression of mRNA

na

compared with the untreated control, respectively. The expression changes are represented with comparisons of untreated control RNA-seq (Fold change = 1.0). Hierarchical clustering

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analysis (HCA) based on Euclidean distances was done using MeV 4.9 to assess the differences/similarities among all four transcriptomes on the 344 genes. (C) Heat map showing expression results of selected genes in different treated macrophages. The heat map displays the enrichment of immune response-related genes of overlapping genes from RNAseq expression data set from treated cells compared with untreated control cells (Fold change = 1.0).

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Authorship contributions Conceptualization and experiment design, Y.-R.S., C.-W.C., and H.-D.H.; Investigation, Y.R.S., A.-R.H., S.-G.P., and H.-D.H.; Data analyses, Y.-R.S., Y.-K.R., and H.-D.H.; writing—

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original draft preparation, Y.-R.S.; writing—review and editing, H.-D.H.

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Highlights  Enzyme-assisted extraction was successfully applied for lotus leaf polysaccharides.  Properties of polysaccharides extracted by different methods were compared.

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 LLEP-P by pectinase assistance exhibited stronger immunostimulatory activities.

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