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. 2025 Feb 13;10(7):7153–7162. doi: 10.1021/acsomega.4c10466

Structural Characterization and Antioxidant Properties of a Novel Polysaccharide Isolated from Gymnopetalum cochinchinense

Trung Hieu Le †, Thi Lan Huong Hoang †,‡, Thi Hong Chuong Nguyen §,∥,*, Thi Van Thi Tran †, Do Thi Thuy Van ⊥,*, Tran Thi Ngoc Bich ⊥, Minh Nhung Nguyen †, Lam Son Le †, Xuan Anh Vu Ho †, Thanh Minh Tran †, Nga Hang Thi Phan #, Chinh Chien Nguyen §,∥,*
PMCID: PMC11865989  PMID: 40028130

Abstract

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In this investigation, a novel heteropolysaccharide, denoted as PS-HW1, is isolated from Gymnopetalum cochinchinense. The achieved PS-HW1 polymer exhibits a molecular weight of 1.22 × 102 kDa and composes the residues of d-glucose, d-fructose, and d-galactose in a 1:1:2 ratio. The structure of PS-HW1 is elucidated via gas chromatography-mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance spectroscopy, revealing a backbone consisting of (1→4)-d-glucopyranose, (1→4)-d-galactopyranose, and (1→3)-d-fructopyranose residues. Furthermore, PS-HW1 demonstrates remarkable antioxidant activity, with low IC50 values of 0.88 and 3.51 mg·mL–1 in the DPPH and ABTS assays, respectively. Its total antioxidant capacity is determined to be 0.2672 ± 0.0042 mg of GA·g–1 or 0.1765 ± 0.0028 mg of AS·g–1. Additionally, PS-HW1 shows significant inhibitory effects on nitric oxide, acetylcholinesterase, and cytotoxicity against HepG2, MCF-7, KB, and SK-LU-1 cancer cells. Such findings emphasize the considerable potential of PS-HW1 from G. cochinchinense for pharmaceutical applications.

1. Introduction

Polysaccharides (PS), monosaccharide-constructed polymeric carbohydrates, have gained increasing attention because of their potential applications in various industrial fields.1 Indeed, the European Polysaccharide Network of Excellence (EPNOE) has identified polysaccharides as an essential component in five key areas as follows: (i) materials and engineering; (ii) food and nutrition; (iii) biomedical applications (e.g., drug delivery systems, wound healing, tissue engineering, anti-inflammatory treatments, antitumors, antioxidants); (iv) chemistry, biology, and physics; and (v) skills and education.2

Polysaccharides exhibit significant potential in existing medical treatments because of their diverse biological activities, excellent biocompatibility, and nontoxic nature.3,4 Additionally, polysaccharides have demonstrated therapeutic benefits in wound healing, immunomodulation, and targeted drug delivery.5 Furthermore, their ability to form hydrogels and nanoparticles makes them well suited for innovative applications in tissue engineering, management of chronic diseases, combating antibiotic resistance, and cancer therapies.4

Antioxidant activities derived from PS have emerged in the context of the fast-paced increase in free-radical-associated health issues over the past decades. Their bioactivities are driven by structural configuration (e.g., monosaccharide composition, bonding manners within main chains, molecular weight, hydroxyl groups, and esterification).6 Yu et al. reported the identification of a polysaccharide possessing fructose-rich macromolecules and a molecular weight of 3.24 × 103 Da. Such a compound demonstrates significant immunomodulatory and antioxidant activities in vivo.7 Notably, the polysaccharide exhibited a high content of galactose and glucose, which contributed to its statistically significant (p < 0.05) antimicrobial activity against selected Gram-negative and Gram-positive bacterial strains. In particular, the galactose component was associated with diverse bioactivities, including anti-inflammatory, antioxidant, antitumor, immunomodulatory, antibacterial, and prebiotic properties.1 To this end, biomedical plants have been considered as the greatest potential PS source, especially taking into account the huge number of those species available in nature.

Gymnopetalum cochinchinense, a creeping herbaceous plant with profuse branching, belongs to the genus Gymnopetalum in the Cucurbitaceae family. This plant has accounted for an essential function in Vietnamese traditional medicine because of its therapeutic effects in treating various conditions (e.g., liver cooling, melasma, detoxification, phlegm expulsion, amenorrhea management, and menstrual pain relief).8,9 Phytochemical studies have demonstrated that this medicinal plant possesses antioxidant properties.9 Additionally, it exhibits a range of biological activities, including antiulcer effects, prevention of leukocyte-related disorders, protection against typhoid, antipyretic activity, anti-inflammatory effects, bronchial dilation, wound healing,10,11 and antibacterial and antifungal properties.9 However, the discovery of PSs and associated biological activities of this plant remains to be elucidated.

The presented study aims to isolate and determine the polysaccharides’ structure from G. cochinchinense. A combination of methylation analysis, gas chromatography (GC) spectrometry, infrared (IR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy has unfolded a novel PS structure (denoted as PS-HW1) for the first time. Additionally, the investigation of the antioxidant activities is unveiled to provide sufficient potential properties of the isolated PS-HW1 polysaccharide.

2. Results and Discussion

2.1. Extraction and Structural Characterizations of PS-HW1

The dried G. cochinchinense plant-containing PS-HW1 polysaccharide was collected via hot water extraction coupled with ethanol precipitation before deproteinization and decolorization. Diethylaminoethyl (DEAE)-cellulose column and gel permeation chromatography (GPC) were conducted to purify and collect the crude PS-W1 polysaccharide12 (see Section 2.2 for experimental details).

Figures 1 and S1 illustrate the high-performance gel permeation chromatography (HPGPC) profile of PS-HW1. The intensity of this peak reaches 1.35 × 102 kDa, and the average molecular mass is estimated to be approximately 1.22 × 102 kDa. The polydispersity index, calculated as the Mw/Mn ratio, is found to be approximately 1.24, signifying the heterostructure characteristic of the achieved PS-HW1 sample.

Figure 1.

Figure 1

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Molecular mass chromatogram of PS-HW1 from G. cochinchinense obtained by HPGPC.

The Fourier transform infrared (FT-IR) spectrum was used to quantitatively investigate the functional groups and characteristic bonds existing in structural PS-HW1, as depicted in Figure 2. The peak observed at approximately 3418 cm–1 corresponds to the stretching vibration of the O–H bond within the polysaccharide structure.13 Bands appearing at around 1634 cm–1 are attributed to surface-bound water. This can be explained by the adhesion of molecules to the polysaccharide surface via hydrogen bonding or van der Waals interactions.14 Strong absorption bands located at 1414, 1329, 1242, 1151, and 960 cm–1 can be assigned to the stretching vibrations of the C–O bond.15 The peaks at 1414 and 1329 cm–1 could be attributed to the C–O bond of β-d-galactose. Additionally, the minor absorption band observed at 960 cm–1 suggests the presence of the C–O bond of fructose residues.16 Bands appearing at 1242 and 1151 cm–1 could be attributed to the C–O bond of α-d-glucose.17−19 More importantly, the peaks at 1151 and 1024 cm–1 could be attributed to glucopyranoside and α-type glucan bonds, respectively.14 These findings from the infrared spectrum reveal distinct features of the polysaccharide composition within PS-HW1.

Figure 2.

Figure 2

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Fourier transform infrared spectroscopy of PS-HW1 from G. cochinchinense.

The subsequent characterization focused on investigating monosaccharide composition and glycosidic bonds by analyzing the acetylated carbon positions in the resulting alditol acetate derivatives using hydrolysis, acetyl derivatization, and GC-flame ionization detector (GC-FID) and GC-mass spectrometry (GC-MS) analysis. As shown in Figure S2, the GC-FID results from alditol acetate derivatives indicate the presence of d-glucose, d-galactose, and d-fructose residues within the polysaccharide backbone. The GC-MS of the PS-W1 sample exhibits the intensive peaks centering at retention times of 19.00, 22.83, and 24.62, corresponding to 1,4,5-tri-O-acetyl-2,3,6-tri-O-methylglucitol, 2,3,5-tri-O-acetyl-1,4,6-tri-O-methylmannitol, and 1,4,5-tri-O-acetyl-2,3,6-tri-O-methylgalactitol, respectively (Figure S3). Additionally, the presence of 4-O-substituted Glcp and Galp, and 3-O-substituted Fruf suggests that pyranose rings contain glucose and galactose, whereas furanose rings consist of fructose. The analyses confirm the existence of three types of glycosidic linkages within PS-HW1 as follows: glucosyl (1→4), galactosyl (1→4), and fructosyl (1→3).

Nuclear magnetic resonance (NMR) spectroscopy was employed to elucidate the structure of the investigated sample in depth. As shown in Figure S4, the characteristic peaks associated with small organic molecules and peptides are absent in the 1H NMR spectrum of the PS-HW1 sample. This implies a high degree of purity in the obtained polysaccharide. Signals centering at δH 5.39 and 4.95 ppm with a ratio of 1:2 are attributed to the presence of anomeric protons. Additionally, signals observed in the chemical shift region of 3.50 and 4.17 ppm can be assigned to sugar protons, as exhibited in Figure 3A. Regarding the full 13C NMR spectrum of the PS-HW1 sample (Figure S5), the peaks appearing at δC 92.1 ppm and δC 103.8 ppm are attributed to C-1 of the α-d-glucopyranosyl moiety and the anomeric carbon region corresponding to C-2 of fructose.20 Furthermore, Figure 3B exhibits two distinct signals at δC 98.4 and 98.0 ppm, which are assigned to the C-1 carbons of β-d-galactose.21 To this end, NMR results, combined with methylation analysis, prove the presence of d-glucose, d-galactose, and d-fructose in the repeating unit of the PS-HW1 structure.

Figure 3.

Figure 3

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One-dimensional (1D) NMR spectra of PS-HW1. (A) 1H NMR and (B) 13C NMR.

The two-dimensional (2D) NMR technique was employed to elucidate the precise chemical structure of the PS-HW1 polymer. The specific carbon and proton positions within the cyclic structure of PS-HW1 could be unveiled through the analysis of the heteronuclear single quantum coherence (HSQC) spectrum in conjunction with 1H NMR and 13C NMR spectra, as shown in Table 1 and Figure S6. Furthermore, the correlated spectroscopy (COSY) spectrum reveals 1H–1H couplings, which provide insight into the connectivity between adjacent protons within the PS-HW1 molecular framework (Table 1, Figure S7). Additionally, the heteronuclear multiple bond correlation (HMBC) spectrum (Figure S8) unveils correlations between protons and adjacent carbons in the PS-HW1 structure. Significant correlations, including those of residues A–D, are presented in Table 1 and Figure 4. These correlations facilitate the determination of bond arrangements between protons and carbons within the saccharide components of PS-HW1, as summarized in detail in Table 2. To this point, the structure of the repeating unit is unambiguously unveiled as [→4)-β-d-Galp-(1→4)-β-d-Galp-(1→4)-α-d-Glucp(1→3)-β-d-Frucf-2→]n, suggesting a novel polysaccharide structure isolated from G. cochinchinense, as illustrated in Figure 5.

Table 1. Correlations Involving Atoms Observed in the HSQC, COSY, and HBMC Spectra of PS-HW1 Were Recorded in D2O.

sugar residue sugar linkage HSQC (1H → 13C) COSY (1H → 1H) HMBC (1H → 13C)
A →4)-β-d-galactopyranoside-(1→ δ 4.95/δ 98.4, δ 3.80/δ 68.5, δ 3.83/δ 69.5, δ 3.68/δ 66.5, δ 4.10/δ 68.8, δ 3.88/δ 69.2 δ 3.80/δ 3.83, δ 3.83/δ 3.80/δ 3.68, δ 3.68/δ 3.83/δ 4.10, δ 4.10/δ 3.68/δ 3.88 δ 4.95/δ 68.5, δ 3.80/δ 69.5, δ 3.83/δ 66.5/δ 68.5, δ 3.68/δ 69.5/δ 68.8, δ 4.10/δ 66.5/δ 69.2
B →4)-β-d-galactopyranoside-(1→ δ 4.95/δ 98.0, δ 3.82/δ 68.3, δ 4.02/δ 65.9, δ 3.71/δ 72.7, δ 3.54/δ 71.0, δ 3.95/δ 69.4 δ 3.82/δ 4.02, δ 4.02/δ 3.82/δ 3.71, δ 3.71/δ 4.02/δ 3.54, δ 3.54/δ 3.71/δ 3.95 δ 4.95/δ 68.3, δ 3.82/δ 65.9, δ 4.02/δ 68.3/δ 72.7, δ 3.71/δ 65.9/δ 71.0, δ 3.54/δ 72.7/δ 69.4
C →4)-α-d-glucopyranoside-(1→ δ 5.39/δ 2.1, δ 3.52/δ 69.5, δ 3.97/δ 71.0, δ 4.01/δ 71.3, δ 3.73/δ 61.2, δ 3.96/δ 69.3 δ 3.52/δ 3.97, δ 3.97/δ 3.52/4.01, δ 4.01/δ 3.97/δ 3.73, δ 3.73/δ 4.01/δ 3.96 δ 5.39/δ 69.5, δ 3.97/δ 69.5/δ 71.3, δ 4.01/δ 71.0/δ 61.2, δ 3.73/δ 71.3/δ 69.3
D →3)-β-d-frucofuranoside-(1→ δ 4.03/δ 74.0, δ 3.64/δ 61.4, δ 4.17/δ 6.4, δ 3.86/δ 81.3, δ 3.75/δ 62.5 δ 4.17/δ 3.64/δ 3.75, δ 3.75/δ 4.17/δ 3.86 δ 4.03/δ 103.8, δ 3.64/δ 103.8/δ 76.4, δ 4.17/δ 61.4, δ 3.75/δ 81.3
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Figure 4.

Figure 4

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HMBC spectrum of PS-HW1.

Table 2. NMR Chemical Shifts (δ, ppm) of PS-HW1 from G. cochinchinense Recorded in D2O.

sugars H-1 H-2 H-3 H-4 H-5 H-6
A →4)-β-d-galactopyranoside-(1→ 4.95 3.80 3.83 3.68 4.10 3.88
B →4)-β-d-galactopyranoside-(1→ 4.95 3.82 4.02 3.71 3.54 3.95
C →4)-α-d-glucopyranoside-(1→ 5.39 3.52 3.97 4.01 3.73 3.96
D →3)-β-d-frucofuranoside-(1→ 4.03   3.64 4.17 3.75 3.86
sugars C-1 C-2 C-3 C-4 C-5 C-6
A →4)-β-d-galactopyranoside-(1→ 98.4 68.5 69.5 66.5 68.8 69.2
B →4)-β-d-galactopyranoside-(1→ 98.0 68.3 65.9 72.7 71.0 69.4
C →4)-α-d-glucopyranoside-(1→ 92.1 69.5 71.0 71.3 61.2 69.3
D →3)-β-d-frucofuranoside-(1→ 74.0 103.8 61.4 76.4 62.5 81.3
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Figure 5.

Figure 5

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Structure of the repeating units of PS-HW1 from G. cochinchinense.

HMBC and nuclear Overhauser effect spectroscopy (NOESY) spectra were conducted to elucidate the structure of the PS-HW1 sugar chain. The HMBC spectrum exhibits notable correlations between protons and carbons, which are observed as follows: A H-1 (δ 4.95) and B C-4 (δ 72.7), B H-1 (δ 4.95) and C C-4 (δ 71.3), and C H-1 (δ 5.39) and D C-3 (δ 61.4). Additionally, the NOESY spectrum further reveals interactions between proton pairs A H-1 (δ 4.95)/B H-4 (δ 3.71), B H-1 (δ 4.95)/C H-4 (δ 4.01), C H-1 (δ 5.39)/D H-3 (δ 3.64), and D H-1 (δ 4.03)/A H-4 (δ 3.68) (Figure 6). Such correlations determine the linkages of the A(1→4)B, B(1→4)C, and C(1→3)D bonds, consistent with GC-MS analysis.

Figure 6.

Figure 6

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1H–1H NOESY spectra of PS-HW1.

2.2. Bioactivity of PS-HW1

2.2.1. Antioxidant Activity

The antioxidant capacity of the achieved PS-HW1 polymer, which has the ability to counteract reactive oxygen species (ROS), was quantified in equivalents of specific antioxidants such as ascorbic acid (AS) and gallic acid. Standard curve equations for gallic acid and ascorbic acid were established, respectively, as follows: Y = 2.0301 × XGA + 0.1127, R = 0.9991 (1), and Y = 4.0026 × XAS – 0.0449, R = 0.9985 (2); where Y represents optical density, XGA represents the concentration of gallic acid, and XAS represents the concentration of ascorbic acid. The total antioxidant capacities (TAC) of the PS-HW1 sample are determined to be 0.2672 ± 0.0042 mg of GA·g–1 and 0.1765 ± 0.0028 mg of AS·g–1, respectively, at the concentration of 1.5 mg·mL–1. Such results signify the outstanding antioxidant properties of PS-HW1 polysaccharide.

Additionally, the ABTS+• and DPPH scavenging activities of PS-HW1 were evaluated. As illustrated in Figure 7, the ABTS+• scavenging activities of PS-HW1 increase from 22.04 to 66.98 %, corresponding to the concentrations range of 1–5 mg·mL–1. Such an activity profile yields an IC50 value of 3.51 mg·mL–1. Similarly, the DPPH radical-scavenging rates of the PS-HW1 sample rise steeply from 18.04 to 81.62 % associated with concentrations of 0.4–2.0 mg·mL–1, resulting in an IC50 value of 0.88 mg·mL–1.

Figure 7.

Figure 7

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Scavenging effects of PS-HW1 on the DPPH radical and the ABTS+• radical.

2.2.2. Cytotoxic Activities

The cytotoxic potential of the PS-HW1 polymer was evaluated on MCF-7, HepG2, SK-LU-1, and KB cancer cell lines. The increased concentrations of PS-HW1 correlate with heightened inhibition of cancer cell proliferation, as shown in Table 3. The PS-HW1 polymer exhibits inhibition rates ranging from 23.24 % to 38.21 % across the tested cancer cell lines at the highest concentration of 500 μg·mL–1, with the highest efficacy observed in the HepG2 cell line. Although the inhibitory effect of PS-HW1 is significantly different from that of the positive control (ellipticine), this compound still demonstrates a notable degree of cytotoxic activity. In other words, PS-HW1 polysaccharides represent a promising approach for the development of therapeutic agents.

Table 3. In Vitro Cytotoxic Activity of PS-HW1 against Various Cancer Cell Lines.
  % inhibition
concentration (μg·mL–1) MCF-7 HepG2 KB SK-LU-1
PS-HW1
500 23.24 ± 1.78 38.21 ± 2.04 25.72 ± 1.69 25.36 ± 1.73
200 11.37 ± 1.03 14.89 ± 1.04 4.42 ± 0.46 7.45 ± 0.77
100 3.59 ± 0.36 10.00 ± 0.86 2.52 ± 0.23 3.60 ± 0.26
50 0.59 ± 0.06 5.04 ± 0.27 1.20 ± 0.11 0.81 ± 0.07
IC50 >500 >500 >500 >500
ellipticine
10 96.70 ± 1.02 95.10 ± 2.02 99.58 ± 2.12 90.56 ± 2.35
2 79.63 ± 1.07 81.10 ± 2.43 85.63 ± 1.18 75.96 ± 1.78
0.4 51.18 ± 1.70 51.32 ± 1.92 51.10 ± 1.37 49.36 ± 1.45
0.08 22.92 ± 1.14 23.27 ± 1.26 23.24 ± 1.39 21.74 ± 1.26
IC50 0.34 ± 0.02 0.33 ± 0.02 0.32 ± 0.02 0.39 ± 0.03
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It can be said that the modest cytotoxic effects of PS-HW1 compared to ellipticine can be attributed to its large molecular weight and structural characteristics, which may hinder cellular uptake and limit direct cytotoxicity, respectively. Future studies could explore chemical modifications to enhance its bioactivity, such as reducing the molecular weight or adding functional groups. Additionally, investigating synergistic effects with other cytotoxic agents may enhance their therapeutic potential.

2.2.3. Acetylcholinesterase (AChE) Inhibitory Activity

Acetylcholinesterase hydrolyzes acetylcholine and other choline esters, which are critical neurotransmitters. Therefore, inhibiting AChE prolongs the action of these neurotransmitters,22 which is beneficial in chronic conditions (e.g., Alzheimer’s disease). In such circumstances, the elevated AChE activity causes an increase in acetylcholine breakdown.23 The result of PS-HW1 indicates a dose-dependent inhibition of AChE, corresponding to a maximum inhibition of 31.31 % at a concentration of 500 μg·mL–1, as presented in Table 4. Therefore, PS-HW1 polymer shows a potential agent as an AChE inhibitor, although this inhibition is lower than that of the standard galanthamine. These findings suggest that PS-HW1 is a valuable adjunctive treatment for Alzheimer’s disease, offering a complementary mechanism to existing therapies and potentially enhancing overall therapeutic outcomes.

Table 4. Results of the Acetylcholinesterase Inhibitory Activity of PS-HW1.
% inhibition AchE
concentration (μg·mL–1) PS-HW1 concentration (μg·mL–1) galanthamine
500 31.31 ± 1.07 10 86.34 ± 1.19
200 16.29 ± 0.75 2 55.16 ± 2.21
100 11.18 ± 0.67 0.4 21.67 ± 1.03
50 2.90 ± 0.30 0.08 7.11 ± 0.56
IC50 >500 IC50 1.80 ± 0.17
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2.2.4. Nitric Oxide NO Inhibitory Activity

This activity evaluation aims to study the impact of PS-HW1 on the intracellular release of nitric oxide in RAW 264.7 macrophages. The results indicate that the PS-HW1 polymer demonstrates a substantial inhibitory effect on NO production. Particularly, PS-HW1 achieves a notable NO inhibition of 37.91 % at a concentration of 500 μg·mL–1, as exhibited in Table 5. Such findings imply that PS-HW1 polysaccharide exhibits moderate potency in attenuating NO production compared to the reference compound, dexamethasone. Consequently, these results highlight the potential pharmacological significance of PS-HW1 in inflammation modulation.

Table 5. Results of the NO Inhibitory Activity of PS-HW1.
% inhibition NO
concentration (μg·mL–1) PS-HW1 concentration (μg·mL–1) dexamethasone
500 37.91 ± 1.98 100 88.02 ± 2.34
200 10.70 ± 1.15 20 53.19 ± 1.02
100 0.76 ± 0.06 4 42.21 ± 0.91
50 –1.40 ± 0.12 0.8 32.66 ± 0.82
IC50 >500 IC50 1.80 ± 0.17
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PS-HW1, a heteropolysaccharide isolated from G. cochinchinense, exhibits remarkable antioxidant, anticancer, and nitric oxide NO inhibitory activity. Structurally, it comprises d-glucose, d-fructose, and d-galactose in a 1:1:2 ratio, connected via glycosidic linkages of (1→4)-d-glucopyranose and (1→3)-d-fructopyranose residues. These structural characteristics underline its bioactivity, as the monosaccharide composition and glycosidic linkages are integral to the biological functions of PS-HW1. Notably, galactose-rich polysaccharides, such as those in Lycium barbarum, are known for their antioxidant and immunomodulatory properties.1 Similarly, d-glucose, reported by Jiang et al. as the main constituent in Boshuzhi-derived polysaccharides, shows significant DPPH free radical-scavenging activity.24 The inclusion of fructose residues enhances solubility and molecular flexibility, promoting efficient interactions with free radicals and biological membranes.25 The (1→4) glycosidic linkage provides structural stability and enzymatic resistance, thereby boosting the bioactivity of the molecule, while the (1→3) linkage improves molecular flexibility, enhancing bioavailability and antioxidant efficacy.25,26 Studies on comparable polysaccharides indicate that glycosidic linkage types are critical determinants of antioxidant and anticancer potential.25,26 PS-HW1, possessing a molecular weight of 1.22 × 102 kDa achieves a favorable balance between solubility and biological activity. Polysaccharides within this molecular weight range are known to demonstrate superior antioxidant and anticancer effects, as they effectively penetrate cellular barriers while retaining the structural complexity required for bioactivity.25 Additionally, the hydroxyl groups in PS-HW1 play a pivotal role in its antioxidant mechanism by donating electrons to neutralize reactive oxygen species (ROS), a property observed in other bioactive polysaccharides.

3. Conclusions

The novel heteropolysaccharide (PS-HW1) isolated from the species G. cochinchinense has been structurally elucidated. PS-HW1 exhibits a molecular weight of 1.22 × 102 kDa and consists of three principal monosaccharides, d-glucose, d-fructose, and d-galactose, arranged in a molar ratio of 1:1:2 within its polymer backbone. Advanced structural characterization techniques, including gas chromatography, infrared spectroscopy, and nuclear magnetic resonance spectroscopy, have identified the repeating structural unit of the polymer as [→4)-β-d-Galp-(1→4)-β-d-Galp-(1→4)-α-d-Glucp(1→3)-β-d-Frucf-2→]n. The total antioxidant capacity of PS-HW1 is determined to be 0.2672 ± 0.0042 mg of GA·g–1 or 0.1765 ± 0.0028 mg of AS·g–1. Notably, PS-HW1 demonstrates significant in vitro antioxidant activity, as evidenced by its low IC50 values of 0.88 and 3.51 mg·mL–1 in the DPPH and ABTS+• assays, respectively. Such results indicate the potential of the PS-HW1 polysaccharide as an effective antioxidant agent. Furthermore, PS-HW1 exhibits inhibition of nitric oxide and acetylcholinesterase and shows cytotoxicity against HepG2, MCF-7, KB, and SK-LU-1 cancer cells. These findings unveil a novel PS-HW1 polysaccharide as a bioactive compound for biomedical applications.

4. Materials and Methods

4.1. Materials, Chemicals, and Equipment

G. cochinchinense plants were collected from Bach Ma, Thua Thien Hue province, Vietnam, and identified by the Department of Biology, College of Sciences, Hue University. A voucher specimen (Code: BM2022.06) was deposited at the Department of Chemistry, Hue University of Sciences, Hue University.

Sephadex G-100, dimethyl sulfoxide ((CH3)2SO, DMSO), dimethyl sulfate ((CH3)2SO4), sodium borohydride (NaBH4), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), gallic acid (GA), ascorbic acid (AS), sodium nitrate, dimethyl sulfoxide (DMSO), and trifluoroacetic acid (TFA) were procured from Sigma-Aldrich Co. Diethylaminoethyl cellulose-52 (DEAE-cellulose-52), H2SO4, ammonium molybdate, sodium phosphate, and a Thermo Fisher Dialysis membrane (Mw cutoff 8000–14,000 Da), were bought from Bum Han Commercial Co. Acetylcholinesterase (AchE), acetylthiocholine Iodid (ACTI), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), sodium nitrite, sulfanilamide, and N-1-naphthyl ethylenediamine dihydrochloride were bought from Sigma Chemical Co. (St. Louis, MO). All utilized reagents and solvents were of analytical grade and strictly adhered to quality standards.

The HepG2, A549, KB, and MCF-7 cancer cell lines were supplied by Long Island University and the University of Milan. These cell lines were cultivated in appropriate culture media supplemented with 10 % fetal bovine serum (FBS) and essential ingredients. Culturing procedures were strictly carried out under sterile conditions with 5 % CO2, maintained at 37 °C and 98 % humidity.

The gas chromatography - Flame Ionization detector (GC-FID) was operated under the following conditions: hydrogen gas, air gas, and nitrogen carrier gas. The injection volume was set to 1 μL, and the probe temperature was maintained at 300 °C. The analytical column used was DB-17HT, measuring 30 m in length, 0.25 mm in internal diameter and 0.15 μm in film thickness.

The gas chromatography-mass spectrometry (GC-MS) parameters were as follows. Helium was the carrier gas at 13 psi, with 1 μL sample volume injected at a 20:1 split ratio and an injection temperature of 250 °C. The temperature was program started at 150 °C, increased by 10 °C per min to 280 °C, and was held for 5 min. The MS conditions included an ionization energy of 70 eV, an interface temperature of 280 °C, a source temperature of 230 °C, and a quadrupole temperature of 150 °C.

The Fourier transform infrared (FT-IR) spectra were recorded by using an IRPrestige-21 spectrometer. A sample was prepared by thoroughly grinding and pressing a mixture of dried polysaccharide (PS) powder (2 mg) and potassium bromide (KBr) powder into pellets with a thickness of 1 mm. The measurements were performed across a spectral range of 4000–400 cm–1 with a resolution of 8 cm–1.

Nuclear magnetic resonance (NMR) spectroscopy of the polysaccharide solution was acquired using a Bruker AM500 FT-NMR spectrometer at 500 MHz for 1H and 125 MHz for 13C nuclei at 353 K. Parameters were set at 1.00 s delay (Dl) and 3.28 s acquisition time (AQ) for 1H NMR, and 2.0 s delay and 1.1 s acquisition time for 13C NMR. Two-dimensional spectra (Heteronuclear Single Quantum Coherence spectroscopy (HSQC), heteronuclear multiple bond correlation (HMBC), and nuclear overhauser enhancement spectroscopy (NOESY)) were used to elucidate sugar residues, with chemical shifts reported in ppm.

4.2. Extraction, Isolation, and Purification of PS-HW1

The extraction, isolation, and purification of PS-HW1 were carried out based on the methods of Do et al.27 and Zhu et al.12 The aerial parts of G. cochinchinense were first air-dried under shade conditions, followed by convection drying at 50 °C for 24 h. The dried material was ground into a fine powder and sieved through a 200-mesh screen to ensure a fine powder. Five hundred grams of dry plant material powder was extracted using ethanol solvent at 78 °C (3 times, 3 h per time) to eliminate pigments and lipids. The material powder was then further extracted with distilled water at 100 °C (3 times, 4 h per time). The resulting extract was concentrated via vacuum evaporation at 60 °C, followed by the addition of 96 % ethanol in a ratio of 1:4 (extract/ethanol, v/v). The mixture was stirred and allowed to stand overnight at −10 °C.27 The resulting precipitate was subjected to freeze-drying and deproteinization using the Sevag method. It was then dissolved in distilled water and filtered through a 0.45 μm membrane filter before loading onto a DEAE-cellulose column (2.6 × 40 cm2). The elution process was carried out using distilled water, followed by a NaCl solution (0.1–0.5 M) at a flow rate of 0.5 mL·min–1. The crude polysaccharide obtained was further purified through gel permeation chromatography (GPC) utilizing a Sephadex G-100 column (1 cm × 50 cm), with deionized water as the mobile phase, flowing at a rate of 0.4 mL·min–1,12 yielding the purification polysaccharide signed as PS-HW1.

4.3. Molecular Mass Estimation

GPC analysis was performed to determine the weighted average molecular weight (Mw), number-average molecular weight (Mn), and polydispersity index (Mw/Mn) of PS-HW1. The purified PS-HW1 was dissolved in a 0.1 M NaNO3 solution (10 μL) and injected into the chromatographic system (Agilent 1100 series coupled with an MS detector; microTOF-QII Bruker). A calibrated Ultrahydrogen 500 column (7.8 mm × 300 mm, 10 μm) was used for elution employing a 0.1 M NaNO3 solution at 40 °C with a flow rate of 1 mL·min–1, while pullulan, with known molecular masses, including 5, 10, 20, 50, 100, 200, 400, and 800 kDa, was used as a standard and specific refractive index (dn/dc) = 0.146 mg·mL–1.28

4.4. Monosaccharide and Linkage Analysis

The analytical methodology was based on the method of Le et al.,28 which began with polysaccharide methylation using dimethyl sulfate and solid sodium hydroxide in DMSO at 60 °C for 16 h. Next, 50 g of methylated PS underwent hydrolysis for monosaccharide analysis using 2 M TFA at 120 °C for 2 h, followed by TFA removal via nitrogen stream evaporation. Partially methylated monosaccharides were converted to alditol acetates via reduction with 0.25 M NaBH4 in NH3, neutralized with 10 % acetic acid in MeOH, and lyophilized to remove boric acid. Acetylation was conducted with a mixture of acetic anhydride and pyridine (1:1, v/v) at 100 °C for 20 min. The as-obtained sample was desiccated under a nitrogen stream and analyzed by GC-FID and GC-MS.

4.5. Antioxidant Activities

4.5.1. Total Antioxidant Capacity (TAC)

The total antioxidation capacity (TAC) of the samples was evaluated using the method outlined by Nair et al.29 A 0.3 mL portion of the sample was combined with a solution containing 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate, making up a total volume of 3 mL. This mixture was then incubated at 95 °C for 90 min. Afterward, the mixture was cooled to 25 °C, and the absorbance was measured using a Jasco V-630 spectrophotometer (Japan Spectroscopic Company, Japan, wavelength range 200–900 nm) at 695 nm against a blank consisting of 3 mL of the reagent solution without any sample. The TAC was determined in gallic acid (GA) and ascorbic acid (AS) equivalents.

4.5.2. DPPH Radical-Scavenging Activity

The DPPH radical-scavenging capacity was evaluated according to the procedure of Le et al.30 Two milliliters of samples dissolved in DMSO at different concentrations (0.2–2.5 mg·mL–1) were mixed with 1 mL of 100 μM DPPH solution in methanol and allowed to react for 30 min at ambient temperature. The absorbance was then measured using UV–vis spectroscopy at a wavelength of 517 nm. Ascorbic acid served as the standard reference compound. The IC50 value assessed the effectiveness of the radical-scavenging activity.

4.5.3. ABTS+• Radical-Scavenging Activity

The ABTS+• radical-scavenging activity was assessed using the ABTS+• radical decolorization method, following the protocol described by Re et al.31 0.1 mL of samples at concentrations ranging from 1 to 5 mg·mL–1 were combined with ABTS+• solution (3.9 mL). The absorbance of the obtained mixtures was measured at 734 nm. Ascorbic acid served as the standard reference. The effectiveness of the ABTS+• scavenging activity was determined by the IC50 value.

4.6. Nitric Oxide (NO) Production Assay

Nitric oxide (NO) production was quantified by measuring nitrite accumulation within the cellular milieu, utilizing the Griess Reagent System.32,33 RAW 264.7 macrophages were seeded at a density of 2 × 105 cells per well in a 96-well plate. After a 2 h pretreatment with various concentrations of test samples (50, 100, 200, and 500 μg·mL–1) at 37 °C, the cells were stimulated with lipopolysaccharide (LPS) at 10 μg·mL–1 for 24 h. Subsequently, 100 μL of the culture supernatant was mixed with an equal volume of Griess reagent (including 50 μL of 1 % sulfanilamide in 5 % phosphoric acid and 50 μL of 0.1 % N-1-naphthyl ethylenediamine dihydrochloride) and incubated at room temperature for 10 min. The optical density at 540 nm was measured using a microplate reader, and NO concentration was determined by referencing a calibration curve constructed with sodium nitrite standards.34 Dexamethasone served as a positive control.

4.7. Anti-Acetylcholinesterase Activity

Acetylcholinesterase (AChE) activity was determined according to the method outlined by Ellman et al.35 The mixture containing 140 μL of 100 mM sodium phosphate buffer (pH 8.0), 20 μL of the sample (polysaccharide dissolved in 100% DMSO and then diluted with distilled water), and 20 μL of AChE solution was incubated for 15 min at 25 °C. Subsequently, 10 μL of 2.5 mM DTNB and 10 μL of 2.5 mM acetylthiocholine iodide were added sequentially. The absorbance of the resulting solution was measured at 412 nm. Galanthamine was used as a positive control.36,37

4.8. Cell Culture and Cytotoxicity Studies

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to evaluate cytotoxicity against HepG2, SK-LU-1, KB, and MCF-7 cancer cell lines according to the procedure of Alley and Skehan et al.38,39 Samples were prepared by diluting with DMSO over a range of concentrations from 50 to 500 μg·mL–1. The percentage of cell growth inhibition was calculated using formula 1, where A(testcell), A(control), and A(blank) represented absorbance readings of the test cell sample, control sample, and blank sample, respectively.

4.8. 1

4.9. Statistical Analysis

All experiments were conducted in triplicate (n = 3) unless specified. Data are expressed as mean values ± standard deviation (SD) or standard error of the mean (SEM). Statistical analysis was performed using Origin 8.0 and Microsoft Excel (2010). Differences with p-values <0.05 were deemed statistically significant.

Acknowledgments

This study was funded by the Ministry of Education and Training Vietnam under project number B2024.DNA.22 (D.T.T.V.).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c10466.

  • Spectrum chromatogram of PS-HW1 from G. cochinchinense obtained by HPGPC; results of GC-FID analysis of alditol acetate derivatives; results of GC-MS analysis of alditol acetate derivatives; the full 1H NMR spectrum of PS-HW1; the full 13C NMR spectrum of PS-HW1; 2D 1H–13C HSQC spectrum of PS-HW1; 1H–1H COSY spectrum of PS-HW1; and the full HMBC spectrum of PS-HW1 (PDF)

Author Contributions

∇ T.H.L. and T.L.H.H. contributed equally. C.C.N. and D.T.T.V. conceived the idea of developing the manuscript. T.H.L., T.L.H.H., and C.C.N. wrote the manuscript and collected all relevant references. T.H.C.N., T.V.T.T., T.T.N.B., M.N.N., L.S.L., X.A.V.H., T.M.T., and T.H.N.P. all contributed in writing and reviewed the manuscript. All authors have approved the final version of the manuscript.

The authors declare no competing financial interest.

Supplementary Material

ao4c10466_si_001.pdf (976.9KB, pdf)

References

  1. Wang Z.; Zheng Y.; Lai Z.; Hu X.; Wang L.; Wang X.; Li Z.; Gao M.; Yang Y.; Wang Q.; Li N. Effect of monosaccharide composition and proportion on the bioactivity of polysaccharides: A review. Int. J. Biol. Macromol. 2024, 254, 127955 10.1016/j.ijbiomac.2023.127955. [DOI] [PubMed] [Google Scholar]
  2. Gericke M.; Amaral A. J. R.; Budtova T.; De Wever P.; Groth T.; Heinze T.; Höfte H.; Huber A.; Ikkala O.; Kapuśniak J.; Kargl R.; Mano J. F.; Másson M.; Matricardi P.; Medronho B.; Norgren M.; Nypelö T.; Nyström L.; Roig A.; Sauer M.; Schols H. A.; van der Linden J.; Wrodnigg T. M.; Xu C.; Yakubov G. E.; Stana Kleinschek K.; Fardim P. The European Polysaccharide Network of Excellence (EPNOE) research roadmap 2040: Advanced strategies for exploiting the vast potential of polysaccharides as renewable bioresources. Carbohydr. Polym. 2024, 326, 121633 10.1016/j.carbpol.2023.121633. [DOI] [PubMed] [Google Scholar]
  3. Wei Z.; Yang J. H.; Liu Z. Q.; Xu F.; Zhou J. X.; Zrínyi M.; Osada Y.; Chen Y. M. Novel Biocompatible Polysaccharide-Based Self-Healing Hydrogel. Adv. Funct. Mater. 2015, 25 (9), 1352–1359. 10.1002/adfm.201401502. [DOI] [Google Scholar]
  4. Yu Y.; Shen M.; Song Q.; Xie J. Biological activities and pharmaceutical applications of polysaccharide from natural resources: A review. Carbohydr. Polym. 2018, 183, 91–101. 10.1016/j.carbpol.2017.12.009. [DOI] [PubMed] [Google Scholar]
  5. Cui M.; Zhang M.; Liu K. Colon-targeted drug delivery of polysaccharide-based nanocarriers for synergistic treatment of inflammatory bowel disease: A review. Carbohydr. Polym. 2021, 272, 118530 10.1016/j.carbpol.2021.118530. [DOI] [PubMed] [Google Scholar]
  6. Wang Y.; Yang Z.; Wei X. Antioxidant activities potential of tea polysaccharide fractions obtained by ultra filtration. Int. J. Biol. Macromol. 2012, 50 (3), 558–564. 10.1016/j.ijbiomac.2011.12.028. [DOI] [PubMed] [Google Scholar]
  7. Yu J.; Dong X.-d.; Jiao J.-s.; Yu S.-s.; Ji H.-y.; Liu A.-j.; Chen Y. Extraction, purification, and biological activities in vivo of a novel fructose-rich polysaccharide from Codonopsis pilosula. Ind. Crops Prod. 2022, 176, 114309 10.1016/j.indcrop.2021.114309. [DOI] [Google Scholar]
  8. De Wilde W.; Duyfjes B. Review of the genus Gymnopetalum (Cucurbitaceae). BLUMEA 2006, 51 (2), 281–296. 10.3767/000651906X622229. [DOI] [Google Scholar]
  9. Kumar S.; Das G.; Shin H. S.; Pradeep Kumar P.; Patra J. K. Evaluation of medicinal values of Gymnopetalum chinense (Lour.) Merr., a lesser known cucurbit from Eastern Ghats of India. Braz. Arch. Biol. Technol. 2017, 60, e17160580 10.1590/1678-4324-2017160580. [DOI] [Google Scholar]
  10. Seal T.; Chaudhuri K.; Pillai B. Antioxidant activity of some selected wild edible fruits of North-eastern region in India and effect of solvent extraction system. Global J. Environ. Res. 2012, 6, 84. [Google Scholar]
  11. Rahmatullah M. A survey of preventive medicinal plants used by the Chakma residents of Hatimara (south) village of Rangamati district, Bangladesh. Am.-Eurasian J. Sustainable Agric. 2011, 5, 92–96. [Google Scholar]
  12. Zhu J.; Tan Z.; Zhang Z.; Shi X. Characterization on structure and bioactivities of an exopolysaccharide from Lactobacillus curvatus SJTUF 62116. Int. J. Biol. Macromol. 2022, 210, 504–517. 10.1016/j.ijbiomac.2022.04.203. [DOI] [PubMed] [Google Scholar]
  13. Zengenni Z.; Yi Y.; Guo Y.; Wang R.; Hu Q.; Xiong X. Chemical characterization and antitumor activities of polysaccharide extracted from Ganoderma lucidum. Int. J. Mol. Sci. 2014, 15, 9103. 10.3390/ijms15059103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kacuráková M.; Capek P.; Sasinkova V.; Wellner N.; Ebringerova A. FT-IR study of plant cell wall model compounds: pectic polysaccharides and hemicelluloses. Carbohydr. Polym. 2000, 43 (2), 195–203. 10.1016/S0144-8617(00)00151-X. [DOI] [Google Scholar]
  15. Wu F.; Yan H.; Ma X.; Jia J.; Zhang G.; Guo X.; Gui Z. Comparison of the structural characterization and biological activity of acidic polysaccharides from Cordyceps militaris cultured with different media. World J. Microbiol. Biotechnol. 2012, 28, 2029–2038. 10.1007/s11274-012-1005-6. [DOI] [PubMed] [Google Scholar]
  16. Zhang M.; Ding R.; Hu F. Comparison on FTIR spectrum and thermal analysis for four types of Rehamnnia glutinosa and their extracts. Res. Sq. 2022, 1–14. [Google Scholar]
  17. Černá M.; Barros A. S.; Nunes A.; Rocha S. l. M.; Delgadillo I.; Čopíková J.; Coimbra M. A. Use of FT-IR spectroscopy as a tool for the analysis of polysaccharide food additives. Carbohydr. Polym. 2003, 51 (4), 383–389. 10.1016/S0144-8617(02)00259-X. [DOI] [Google Scholar]
  18. Nikonenko N. A.; Buslov D. K.; Sushko N. I.; Zhbankov R. G. Investigation of stretching vibrations of glycosidic linkages in disaccharides and polysaccarides with use of IR spectra deconvolution. Biopolymers 2000, 57 (4), 257–262. 10.1002/1097-0282(2000)57:4<257::AID-BIP7>3.0.CO;2-3. [DOI] [PubMed] [Google Scholar]
  19. Zhbankov R. G.; Andrianov V. M.; Marchewka M. K. Fourier transform IR and Raman spectroscopy and structure of carbohydrates. J. Mol. Struct. 1997, 436–437, 637–654. 10.1016/S0022-2860(97)00141-5. [DOI] [Google Scholar]
  20. Tajima K.; Uenishi N.; Fujiwara M.; Erata T.; Munekata M.; Takai M. The production of a new water-soluble polysaccharide by Acetobacter xylinum NCI 1005 and its structural analysis by NMR spectroscopy. Carbohydr. Res. 1997, 305 (1), 117–122. 10.1016/S0008-6215(97)00264-4. [DOI] [PubMed] [Google Scholar]
  21. Das D.; Mondal S.; Roy S. K.; Maiti D.; Bhunia B.; Maiti T. K.; Islam S. S. Isolation and characterization of a heteropolysaccharide from the corm of Amorphophallus campanulatus. Carbohydr. Res. 2009, 344 (18), 2581–2585. 10.1016/j.carres.2009.09.025. [DOI] [PubMed] [Google Scholar]
  22. Strelnik A. D.; Petukhov A. S.; Zueva I. V.; Zobov V. V.; Petrov K. A.; Nikolsky E. E.; Balakin K. V.; Bachurin S. O.; Shtyrlin Y. G. Novel potent pyridoxine-based inhibitors of AChE and BChE, structural analogs of pyridostigmine, with improved in vivo safety profile. Bioorg. Med. Chem. Lett. 2016, 26 (16), 4092–4094. 10.1016/j.bmcl.2016.06.070. [DOI] [PubMed] [Google Scholar]
  23. Lehninger A. L.; Nelson D. L.; Cox M. M.. Lehninger Principles of Biochemistry; Macmillan, 2005. [Google Scholar]
  24. Jiang J.; Kong F.; Li N.; Zhang D.; Yan C.; Lv H. Purification, structural characterization and in vitro antioxidant activity of a novel polysaccharide from Boshuzhi. Carbohydr. Polym. 2016, 147, 365–371. 10.1016/j.carbpol.2016.04.001. [DOI] [PubMed] [Google Scholar]
  25. Liu J.; Willför S.; Xu C. A review of bioactive plant polysaccharides: Biological activities, functionalization, and biomedical applications. Bioact. Carbohydr. Diet. Fibre 2015, 5 (1), 31–61. 10.1016/j.bcdf.2014.12.001. [DOI] [Google Scholar]
  26. Li S.; Xiong Q.; Lai X.; Li X.; Wan M.; Zhang J.; Yan Y.; Cao M.; Lu L.; Guan J.; Zhang D.; Lin Y. Molecular Modification of Polysaccharides and Resulting Bioactivities. Compr. Rev. Food Sci. Food Saf. 2016, 15 (2), 237–250. 10.1111/1541-4337.12161. [DOI] [PubMed] [Google Scholar]
  27. Do T. B. T.; Tran B. K.; Tran T. V. T.; Le T. H.; Cnockaert M.; Vandamme P.; Nguyen T. H. C.; Nguyen C. C.; Hong S. H.; Kim S. Y.; Van Le Q. Decoding the capability of Lactobacillus plantarum W1 isolated from soybean whey in producing an exopolysaccharide. ACS Omega 2020, 5 (51), 33387–33394. 10.1021/acsomega.0c05256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Le T. H.; Thi Tran T. V.; Tran V. K.; Vu Ho X. A.; Tran T. M.; Chau Nguyen D. G.; Chuong Nguyen T. H.; Varma R. S.; Trinh T. K.; Ho T.-T.; et al. Structural characterization of mannoglucan isolated from Ophiocordyceps sobolifera and its antioxidant activities. ACS Omega 2022, 7 (11), 9397–9405. 10.1021/acsomega.1c06651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nair V. D.; Panneerselvam R.; Gopi R. Studies on methanolic extract of Rauvolfia species from Southern Western Ghats of India–In vitro antioxidant properties, characterisation of nutrients and phytochemicals. Ind. Crops Prod. 2012, 39, 17–25. 10.1016/j.indcrop.2012.02.006. [DOI] [Google Scholar]
  30. Le T. H.; Nguyen T. H. C.; Le L. S.; Nguyen D. G. C.; Tran T. V. T.; Nguyen M. N.; Tran T. M.; Bui V. T.; Nguyen T. T. H.; Hoang T. L. H.; Nguyen C. C. Unfolding the structure of polysaccharide isolated from Myxopyrum smilacifolium leaves toward a robust antioxidant. Bioact. Carbohydr. Diet. Fibre 2023, 29, 100347 10.1016/j.bcdf.2023.100347. [DOI] [Google Scholar]
  31. Re R.; Pellegrini N.; Proteggente A.; Pannala A.; Yang M.; Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biol. Med. 1999, 26 (9–10), 1231–1237. 10.1016/S0891-5849(98)00315-3. [DOI] [PubMed] [Google Scholar]
  32. Liao H.; Banbury L.; Liang H.; Wang X.; Lü X.; Hu L.; Wu J. Effect of Honghua (Flos Carthami) on nitric oxide production in RAW 264.7 cells and α-glucosidase activity. J. Tradit. Chin. Med. 2014, 34 (3), 362–368. 10.1016/S0254-6272(14)60103-5. [DOI] [PubMed] [Google Scholar]
  33. Combet S.; Balligand J.-L.; Lameire N.; Goffin E.; Devuyst O. A specific method for measurement of nitric oxide synthase enzymatic activity in peritoneal biopsies. Kidney Int. 2000, 57 (1), 332–338. 10.1046/j.1523-1755.2000.00839.x. [DOI] [PubMed] [Google Scholar]
  34. Tsai P.-J.; Tsai T.-H.; Yu C.-H.; Ho S.-C. Comparison of NO-scavenging and NO-suppressing activities of different herbal teas with those of green tea. Food Chem. 2007, 103 (1), 181–187. 10.1016/j.foodchem.2006.08.013. [DOI] [Google Scholar]
  35. Ellman G. L.; Courtney K. D.; Andres V. Jr; Featherstone R. M. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7 (2), 88–95. 10.1016/0006-2952(61)90145-9. [DOI] [PubMed] [Google Scholar]
  36. Sramek J. J.; Frackiewicz E. J.; Cutler N. R. Review of the acetylcholinesterase inhibitor galanthamine. Expert Opin. Invest. Drugs 2000, 9 (10), 2393–2402. 10.1517/13543784.9.10.2393. [DOI] [PubMed] [Google Scholar]
  37. Sierra K.; de Andrade J. P.; R Tallini L.; Osorio E. H.; Yañéz O.; Osorio M. I.; Oleas N. H.; García-Beltrán O.; de S Borges W.; Bastida J.; Osorio E.; Cortes N. In vitro and in silico analysis of galanthine from Zephyranthes carinata as an inhibitor of acetylcholinesterase. Biomed. Pharmacother. 2022, 150, 113016 10.1016/j.biopha.2022.113016. [DOI] [PubMed] [Google Scholar]
  38. Alley M. C.; Scudiero D. A.; Monks A.; Hursey M. L.; Czerwinski M. J.; Fine D. L.; Abbott B. J.; Mayo J. G.; Shoemaker R. H.; Boyd M. R. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res. 1998, 48 (3), 589–601. [PubMed] [Google Scholar]
  39. Skehan P.; Storeng R.; Scudiero D.; Monks A.; McMahon J.; Vistica D.; Warren J. T.; Bokesch H.; Kenney S.; Boyd M. R. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 1990, 82 (13), 1107–1112. 10.1093/jnci/82.13.1107. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

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