Partial structural characterization, as well as immunomodulatory and anti-aging activities of CP2-c2-s2 polysaccharide from Cordyceps militaris

Abstract

Cordyceps militaris, a typical Chinese traditional medicine, and its polysaccharide in functional food is very promising. The aim of this study was to clarify the structural characterization and biological activity of CP2-c2-s2 from C. militaris (CMP). High-performance gel permeation chromatography (HPGPC) and polyacrylamide gel electrophoresis (PAGE) analysis showed that CP2-c2-s2 was a homogeneous polysaccharide with an average molecular weight of 20 200 Da. The Fourier transform infrared spectroscopy (FTIR) and periodate oxidation analysis revealed that CP2-c2-s2 is a β-pyran polysaccharide, probably with 1→2, 1→4, and 1→6 glycosyl linkages. Micromorphologic observation showed different aggregates and networks from 5 μg mL⁻¹ to 250 μg mL⁻¹. CP2-c2-s2 significantly stimulated the proliferation of T and B lymphocytes, and obviously extended the average lifespan without adverse effects on fecundity, and delayed the age-related decrease in Caenorhabditis elegans. Overall, our results revealed significant immunomodulatory and anti-aging activities of CMP, which may provide useful information for CMP in functional foods.

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1. Introduction

Cordyceps militaris (C. militaris), a rare caterpillar fungus, has similar pharmacologic properties to the well-known Chinese traditional medicine Cordyceps sinensis.¹ Aside from its use as a crude drug, it has been extensively used as a tonic or invigorant since ancient times.² Various constituents, such as polysaccharide, cordycepin, cordycepic acid, and superoxide dismutase (SOD), have shown pronounced biological effects on the human body.³ The medicinal benefits of Cordyceps polysaccharides have attracted increasing research interest. In recent years, many studies have reported the activity, composition, and the physiologic and pharmacologic properties of C. militaris extract.⁴ Previous studies on polysaccharides from various Cordyceps species have demonstrated many interesting biological activities, including anti-tumor activity,⁵ antiviral activity,⁶ immunopotentiation,⁷ hypoglycemic activity,⁸ antioxidant activity,⁹ anti-aging,¹⁰ and hypocholesterolemic activity.¹¹

C. militaris is also widely used as a tonic or an invigorant for longevity in Chinese folk medicine. Experiments on mice showed that polysaccharides from cultivated C. militaris (CMP) fruiting bodies protected mitochondria by scavenging reactive oxygen species (ROS), inhibiting mitochondrial swelling, and increasing antioxidase activity. Therefore, CMP may have pharmaceutical value for mitochondrial protection and anti-aging physiological properties.¹⁰ Although C. militaris has been used in traditional Chinese medicine for millennia, convincing evidence for its anti-aging activities is still lacking. The nematode Caenorhabditis elegans (C. elegans) provides a useful model system for studying organismal aging because of its relatively short lifespan and well-established genetic pathways, which are common traits of diverse species including mammals.¹² Polysaccharides from Chinese traditional herbal medicines, including reishi,¹³ Bletilla striata,¹⁴ Astragalus,¹⁵ and Panax notoginseng,¹⁶ have been shown to extend the lifespan of C. elegans. To determine whether CMP intervenes in the aging process at the organismal level, we investigated the effects of CMP on the anti-aging activity in C. elegans.

Based on previous studies, the polysaccharide CP2-c2-s2 was isolated and purified from C. militaris,¹⁷ but the structure and bioactivity are still unknown. The first objective of this study was to analyze the partial structural characterization by chemical and instrumental analysis, such as high-performance gel permeation chromatography (HPGPC), polyacrylamide gel electrophoresis (PAGE), Fourier transform infrared spectroscopy (FTIR), periodate oxidation, and atomic force microscopy (AFM). The second objective looked into the effects of different CP2-c2-s2 concentrations on the proliferation of mouse spleen lymphocytes, specifically T and B lymphocytes, and determined the anti-aging activity of CMP utilizing the C. elegans model. The immunostimulatory and anti-aging activities were investigated to provide evidence for the development and application of functional foods or polyacrylamide drugs.

2. Materials and methods

2.1. Materials

Powdered C. militaris fruiting bodies were donated by Infinitus (China) Co., Ltd. Male BALB/c mice (6 to 8 weeks old) were purchased from the SPF Laboratory Animal Center at Southern Medical University. Animals were maintained in accordance with the applicable portions of the Animal Welfare Act, and approved by the Animal Ethical Committee of South China Agricultural University, Guangzhou, China. The lentinan injection was from Jinling Pharmaceutical Co., Ltd. The following solvents were used in cell-bases assay: Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), 100× penicillin and streptomycin, non-essential amino acid, 0.25% trypsin with ethylenediaminetetraacetic acid (EDTA) were purchased from Thermo Fisher Scientific Inc. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulphoxide (DMSO), concanavalin A (ConA), lipopolysaccharide (LPS), and dextran standard were purchased from Sigma-Aldrich Co., Ltd (St. Louis, MO, USA). Experimental C. elegans (Bristol strain N2) was obtained from the Caenorhabditis Genetics Center (CGC, University of Minnesota, Minneapolis, MN, USA). The uracil mutant Escherichia coli OP50 (E. coli OP50) was provided by the College of Resource and Environment, South China Agricultural University. All the other chemicals were of analytical reagent grade.

2.2. Preparation of polysaccharide CP2-c2-s2

Crude polysaccharides (CP) were extracted from powdered C. militaris fruiting bodies through sequential hot water extraction, ethanol precipitation, and zinc sulfate deproteination according to the previous report.¹⁷ The CP was fractionated using membrane separation, purified via DEAE-cellulose-52 exchange chromatography, and further purified through Sephadex G-100 gel filtration chromatography.¹⁷ The molecular weights of the polysaccharides were determined using HPGPC, which can characterize molecular weight distribution.

2.3. Homogeneity and molecular weight determination

The homogeneity and molecular weight of CP2-c2-s2 was determined through HPGPC.¹⁸ A Waters 1525 high-performance liquid chromatography (HPLC) system (Waters, Boston, USA) equipped with a Waters ultrahydrogel 1000 column (Waters, Boston, USA) and a Waters 2414 differential refraction detector (Waters, Boston, USA) was used. The operating conditions were as follows: mobile phase, 0.02 mol L⁻¹ potassium dihydrogen phosphate; flow rate, 0.6 mL min⁻¹; column temperature, 35 °C; injection volume, 20 μL; and run time, 45 min. Dextran standards with varying molecular weights T-5200, T-11 600, T-23 800, T-48 600, T-148 000, T-273 000, T-410 000, T-670 000, and T-1 400 000 were prepared at 2 mg mL⁻¹, and their retention times were plotted against the logarithms of their respective molecular weights. The CP2-c2-s2 was dissolved in the mobile phase at a final concentration of 2 mg L⁻¹. The CP2-c2-s2 solution (20 μL) was injected in each run after filtration through a 0.45 μm membrane. The molecular weight was estimated by referring to a calibration curve.

The homogeneity of the purified CP2-c2-s2 was also determined using PAGE.¹⁹ The separating gel solution was 8% and the stacking gel solution was 3.5%. CP2-c2-s2 (1 mg) was diluted to a final volume of 0.1 mL using boric acid buffer, and 10 μL of the sample solution was injected into the sample well. The samples were initially electrophoresed at 80 V for 30 min, and then at 110 V for 1 h. The gels were rinsed with the rinse solution and were oxidized with periodic acid oxidant for 10 min. The gels were then rinsed with sodium metabisulfite solution and stained for 30 min with the Schiff staining solution. The gels were destained three times for 5 min and were scanned using a Universal Hood II (Bio-rad Company, USA) gel imaging analysis system.

2.4. Periodate oxidation

The polysaccharide CP2-c2-s2 (5 mg) was dissolved in 0.015 mol L⁻¹ sodium periodate (5 mL) and kept in the dark at 7 °C. 30 μL aliquots were then withdrawn at 6 h intervals, diluted to 4 mL with distilled water, and absorbance at 223 nm was recorded on a spectrophotometer. Complete oxidation, indicated by a stable absorbance, was reached within 56 h. HIO₄ consumption was measured spectrophotometrically, and formic acid production was determined by titration with 0.005 M NaOH. Glycol (0.2 mL) and phenolphthalein (0.2 mL) were added, and then the periodate oxidation assay was completed.^18a,20

2.5. Fourier transform infrared spectroscopy (FTIR)

The chromatographically purified CP2-c2-s2 was ground with potassium bromide powder and then pressed into pellets for Nexus FTIR (Nicolet Nexus, Thermo Nicolet Company, USA) with a DTGS KBr detector. The scan range was from 4000 to 500 cm⁻¹ at a resolution of 4 cm⁻¹ and the number of scans was 32.^18b

2.6. Atomic force microscope (AFM)

CP2-c2-s2 was observed under an AFM (Nano Scope IIIa; Veeco, USA) in tapping mode according to the reported method.²¹ Stock solution (1.0 mg mL⁻¹) was prepared by adding CP2-c2-s2 into double distilled H₂O. The solution was diluted to final concentrations of 5, 50, 150, and 250 μg mL⁻¹. Approximately 2 μL of diluted CP2-c2-s2 solution was dropped onto the surface of the mica sample carrier, allowed to dry in air, and then observed at room temperature. The tape of the tip was 1 ohm cm to 10 ohm cm THOSTHORUS (n) DOPED Si and the cantilever was RTEST (T: 3.0 μm to 4.5 μm, L: 115 μm to 135 μm, W: 30 μm to 40 μm). The scanning frequency was 1.0 Hz.

2.7. Isolation and culture of mice spleen cells

Splenic cells were harvested from the mice using a core of disposable glass syringe extrusion that was grinded and mixed with 0.87% NH₄Cl buffer. The solution was repeatedly washed with Hank's buffer. Finally, the cells were counted, and the final cell concentration was adjusted to 1.0 × 10⁷ to 1.2 × 10⁷ cells per mL with RPMI 1640 medium (containing 10% FBS, 1% non-essential amino acid, and 1% penicillin-streptomycin.). Splenic cell suspensions were prepared under aseptic conditions.²²

2.8. Splenocyte proliferation assay

Mononuclear cell suspensions (50 μL) were distributed on 96-well plates and cultured for 4 h at 37 °C under a humidified 5% CO₂ atmosphere in an incubator. Each well was treated with 25 μL of the polysaccharide (6.25, 12.5, 25, 50, 100, 125, 200, 250 μg mL⁻¹, final concentration). The positive controls were treated with 25 μL of ConA (final concentration 10 μg mL⁻¹) or LPS (final concentration 10 μg mL⁻¹). However, the blank control was treated with 25 μL of the medium. The plates were then incubated for 48 h. MTT (10 μL) was added to the plates, which were then incubated for another 4 h. A total of 150 μL of DMSO was added to the culture, and the suspension was homogenized for 10 min to fully dissolve the colored material. The absorbance at 490 nm was measured on an ELISA reader. Each experiment was performed in quadruplicate. The following formula was used to calculate the stimulation index (SI): SI = (OD_samples − OD_blank)/(OD_positive − OD_blank).²³

2.9. Preparation of treatment plates

The compounds were dissolved in sterile water and diluted to working concentrations (50, 150, and 300 μg mL⁻¹ CP2-c2-s2; 300 μg mL⁻¹ lentinan; and 50 μg mL⁻¹ acetic acid). Then, 5 μL of the diluted compound was added to the surface of a dry nematode growth medium (NGM) plate spotted with 95 μL of concentrated E. coli OP50. The plates were stored at 4 °C.

2.10. Lifespan assay

C. elegans were routinely propagated at 20 °C on NGM plates in a temperature-controlled incubator with E. coli OP50 as a food source as previously described.²⁴ All the experiment's treatments started from the L4 stage after synchronization according to the previous report.²⁵ All the L4 larvae were transferred into NGM agar hatching plates with or without the treatments, with 60 nematodes for every treatment, and 15 nematodes per plate. The nematodes were transferred into fresh treatment plates every 2 days until progeny production ceased to prevent self-progeny. The nematodes that crawled up the sides of the Petri dishes and died were considered lost and excluded from the analysis. A total of two to three independent trials were performed per treatment.

2.11. Measurement of age-related changes in physiological processes

Body movement were determined as described previously.²⁶ The treated and control nematodes (L4 hermaphrodites, n = 10, each) were placed on individual plates for the egg-laying assay. The nematodes were transferred every 24 h into fresh egg-laying plates until egg laying ceased. The eggs were allowed to hatch at 20 °C for 24 h, and the progeny was counted. Pharyngeal pumping rate was determined by directly counting pharyngeal contractions using a hand counter under 100× magnification. The pumping rate was determined every 2 days using at least 15 animals from each of the treatment groups, and from the untreated controls. Worms that showed no pumping or erratic pumping were excluded.²⁵ Assay for each experiment was performed in triplicate.

2.12. Statistical analysis

All data were expressed as mean ± S.D. The experiments were done three times or as otherwise indicated. Statistical analysis was performed using one-way ANOVA. Differences with p-values less than 0.05 were considered significant.

3. Results and discussion

3.1. Homogeneity and molecular weight of CP2-c2-s2

The HPGPC profile showed a single and symmetrical peak (Fig. 1A), which indicates that CP2-c2-s2 is a homogeneous polysaccharide. The retention time of CP2-c2-s2 was 26.20 min. Based on the calibration curve with dextran standards, the average molecular weight of CP2-c2-s2 was 20 200 Da. The second peak on the spectrum was a solvent peak. The electrophoretogram (Fig. 1B) showed a single band, which confirms that CP2-c2-s2 is homogeneous. The PAGE result was consistent with that of the HPGPC.

Fig. 1 Structural features of CP2-c2-s2. CP2-c2-s2 was investigated using HPGPC (A) and PAGE (B). For HPGPC, a Waters 1525 HPLC system equipped with a Waters ultrahydrogel 1000 column and a Waters 2414 differential refraction detector was used. The operating conditions were as follows: mobile phase, 0.02 mol L⁻¹ potassium dihydrogen phosphate; flow rate, 0.6 mL min⁻¹; column temperature, 35 °C; injection volume, 20 μL; run time, 45 min. For PAGE, the separating and stacking gel solution was 8% and 3.5%, respectively. The samples were initially electrophoresed at 80 V for 30 min, and then at 110 V for 1 h. The gels were rinsed, oxidized, rinsed, stained, destained, and scanned according to the Material and methods 2.3.

3.2. Periodate oxidation analysis

The result of periodate oxidation assay of CP2-c2-s2 was maintained in equilibrium for 56 h. Based on the calibration curve of periodate, periodate consumption and formic acid production were 1.42 × 10⁻² mmol mg⁻¹ polysaccharide and 7.5 × 10⁻⁴ mmol mg⁻¹ polysaccharide, respectively. Formic acid was formed after periodate oxidation of CP2-c2-s2, which indicates that 1→6 glycosyl linkage was probably present in the polysaccharide chain.^18a The periodate consumption was extremely higher than the two-fold amount of formic acid, which indicates the presence of numerous glycosyl linkages, i.e. 1→2 and 1→4 glycosyl linkages, consuming periodate while producing no formic acid.²⁰

3.3. FTIR spectroscopy

The FTIR spectrum was recorded in absorbance mode from 4000 cm⁻¹ to 500 cm⁻¹ to determine the functional groups in the purified CP2-c2-s2 (Fig. 2). The bands in the 3400 cm⁻¹ region were caused by O–H stretching, which indicates the presence of intermolecular and intramolecular carbohydrate hydrogen bonds in the samples. The bands in the 2800 cm⁻¹ to 3000 cm⁻¹ and 1200 cm⁻¹ to 1400 cm⁻¹ regions were caused by C–H stretching and angular vibration, which indicate that CP2-c2-s2 is a carbohydrate. The absorption peak at around 1640 cm⁻¹ showed a characteristic C=C bond, which indicates that CP2-c2-s2 could be composed of uronic acid groups.²⁷ The strong absorption peak at around 1073 cm⁻¹ showed the stretching vibration of C–O of intramolecular C–O–C ring ether, which is the characteristic absorption peak of the pyranose carbohydrate ring. Two types of end carbon–glucoside bonds, α and β, which can be distinguished by FTIR. In the FTIR spectra, the α-type C–H bond has an absorption peak near 844 cm⁻¹, whereas that of the β-type C–H bond is near 891 cm⁻¹.²⁷ The weak absorption peak at around 889 cm⁻¹ indicates the presence of β-glycosidic bond type pyranose in the polysaccharide. CP2-c2-s2 contained hydroxyl groups and a monosaccharide with a pyranoid ring. Therefore, CP2-c2-s2 is a β-pyran polysaccharide.

Fig. 2 FTIR spectrum of polysaccharide CP2-c2-s2. CP2-c2-s2 was ground with KBr powder and then pressed into pellets for Nexus FTIR with a DTGS KBr detector. The scan range was from 4000 to 500 cm⁻¹ at a resolution of 4 cm⁻¹ and the number of scans was 32.

3.4. Atomic force microscopy of CP2-c2-s2

Fig. 3 presented CP2-c2-s2 images obtained when 5 μg mL⁻¹ to 250 μg mL⁻¹ of CP2-c2-s2 solution were used for AFM. At low concentrations (5 μg mL⁻¹), the CP2-c2-s2 molecules formed small dispersed aggregates with different sizes (Fig. 3A). Fig. 3B (50 μg mL⁻¹) clearly formed rod-like helical network structures. The diameter was approximately 100 nm to 150 nm, and the length was about 300 nm to 400 nm, which indicates that the degree of molecular cross-linking and the acting force increased with increasing CP2-c2-s2 concentration. When egg deposition was made at a slightly higher polymer concentration (150 μg mL⁻¹), a big ball-like aggregate structure with a diameter of 150 nm to 400 nm and a height of 30 nm was formed (Fig. 3C). Fig. 3D shows the CP2-c2-s2 micrographs at a higher CP2-c2-s2 concentration (250 μg mL⁻¹) applied onto mica. A strong 3D large network structure appeared on the image, and the diameter was much bigger than that at 50 μg mL⁻¹. Thus, the aggregates and networks were the stable structures of CP2-c2-s2. The information provided by periodate oxidation analysis and FTIR spectroscopy indicated that CP2-c2-s2 contained a large number of branched chains, as well as intramolecular and intermolecular hydrogen bonds, which tended to interact. The network structures were probably formed by small aggregates or the connections of branched chains.

Fig. 3 Atomic force micrographs of CP2-c2-s2. The CP2-c2-s2 concentrations were (A) 5 μg mL⁻¹; (B) 50 μg mL⁻¹; (C) 150 μg mL⁻¹; and (D) 250 μg mL⁻¹. (a) Ichnography; (b) three-dimensional image. The results were obtained with an atomic force microscope in tapping mode. The tape of the tip was 1 ohm cm to 10 ohm cm THOSTHORUS (n) DOPED Si and the cantilever was RTEST (T: 3.0 μm to 4.5 μm, L: 115 μm to 135 μm, W: 30 μm to 40 μm). The scanning frequency was 1.0 Hz.

The shapes of polysaccharides are much more complex than those of nucleotides and proteins. AFM, the technique of choice for conducting single molecule force spectroscopy, is used to study the characteristics of biomacromolecules. In recent years, some reports described the use of AFM to determine the molecular structure of polysaccharides.²² Chen et al. reported that the TPC3-1 molecule from green tea (Camellia sinensis L.) has the anomeric carbon signs of both α and β configurations and highly branched chains.¹⁹ The molecular structure of an acidic polysaccharide, AMBG, of Mesona blumes gum at different concentrations was observed by AFM. AMBG showed spherical lumps at 1 μg mL⁻¹ but an irregular worm-like shape at 10 μg mL⁻¹, which indicated that the viscosity of AMBG might be caused by its notable molecular aggregation.^18a In this study, we initially studied the images of C. militaris polysaccharide CP2-c2-s2 at different concentrations. Different results were observed when CP2-c2-s2 was used at different concentration, and this may have been caused by the composition of CP2-c2-s2, which contains carboxyl and hydroxyl groups.

3.5. Effect of CP2-c2-s2 on spleen lymphocyte proliferation

To ensure that the target immune cells were activated by CP2-c2-s2, the immunomodulatory activity of CP2-c2-s2 at the cellular level was investigated. The effect of CP2-c2-s2 on the proliferation of splenic lymphocytes is shown in Fig. 4. CP2-c2-s2 greatly stimulated the proliferation of lymphocytes, T lymphocytes and B lymphocytes (Fig. 4A–C). Moreover, CP2-c2-s2 showed remarkable proliferative activity on T and B lymphocytes at 12.5 μg mL⁻¹ to 50 μg mL⁻¹. The SI of 50 μg mL⁻¹ CP2-c2-s2 in ConA-induced T lymphocytes and LPS-induced B lymphocytes was 2.5 and 1.2, respectively. However, the proliferation of T and B lymphocytes gradually decreased when the concentration was increased to 100 μg mL⁻¹ to 250 μg mL⁻¹ (Fig. 4B and C). As observed in Fig. 4D, CP2-c2-s2 and lentinan at 50 μg mL⁻¹ significantly enhanced the proliferation of T and B lymphocytes to some extent in vitro. The SI of the B lymphocytes was much higher than that of T lymphocytes. The effect of CP2-c2-s2 on T and B lymphocyte proliferation was much higher than that of lentinan.

Fig. 4 Effects of CP2-c2-s2 on spleen lymphocytes simulation index (SI): (A) polysaccharide stimulation alone, (B) ConA-induced T lymphocytes (10 μg mL⁻¹), (C) LPS-induced B lymphocytes (10 μg mL⁻¹). The concentration of CP2-c2-s2 and lentinan were both 50 μg mL⁻¹ (D). After desired treatment, cell viability was measured by MTT assay as described under Materials and methods 2.8. Each experiment was performed in quadruplicate. The values are presented as means ± SD, n = 5. *Indicates significant difference compared to that of positive control.

Polysaccharides represent a structurally diverse class of macromolecules and their structural variability profoundly affects their cell specificity and biological activity in B cells, T cells, and macrophages.²⁸ CP2-c2-s2 from C. militaris had typical immunostimulatory activity. It activated T lymphocytes and B lymphocytes, and its effect on the LPS-induced proliferation was higher than that on the ConA-induced proliferation. CP2-c2-s2 showed better immune bioactivity than lentinan at the same concentration. The same results were obtained with various Cordyceps polysaccharides. Two acidic polysaccharides CM-jd-CPS2 and CM-jd(Y)-CPS2 from C. militaris exhibited dose-dependent mitogenic effects on mouse splenocytes, and synergistically promoted murine T and B lymphocytes induced by Con A and LPS.^4c C. militaris polysaccharides (CMP) significantly increased the spleen and thymus indices, and enhanced spleen lymphocyte activity and macrophage function.^7a Cordysinocan from C. sinensis activated immune responses in cultured T lymphocytes and macrophages by inducing the phosphorylation of extracellular signal-regulated kinases, and by increasing the phagocytic activity and the enzymatic activity of acid phosphatase.²⁹ Macrophage activation by C. militaris seemed to occur by activating NF-κB and all three MAPKs pathways through dectin-1 and TLR2 macrophage receptors.³⁰ The administration of CMP improved the immune function in mice by significantly increasing the spleen and thymus indices along with the spleen lymphocyte activity, the total quantity of white blood cells, and IgG function in mice serum.^4e

3.6. Anti-aging activity

Compared with the control group, the lifespan curve of C. elegans in the treatment groups all exhibited a slight shift to the right, which indicates that the treatments extended the average lifespan of nematodes (Fig. 5A). The 150 μg mL⁻¹ CP2-c2-s2 (26.72 days), 300 μg mL⁻¹ CP2-c2-s2 (25.18 days), and 300 μg mL⁻¹ lentinan (25.33 days) treatments significantly increased the nematode lifespan compared with the control (22.92 days). The 150 μg mL⁻¹ CP2-c2-s2 treatment resulted in 16.58% longer average lifespan than the control, and its effect was much greater than that of lentinan.

Fig. 5 Effects of CP2-c2-s2, acetic acid, and lentinan on the lifespan (A), body movement (B), pharyngeal pumping rates (C) and fecundity (D) of C. elegans. J50, J150, and J300 indicate 50 μg mL⁻¹, 150 μg mL⁻¹, and 300 μg mL⁻¹ CP2-c2-s2, respectively. AcOH indicates 50 μg mL⁻¹ acetic acid (positive control), X indicates 300 μg mL⁻¹ lentinan, and C indicates the blank control. Nematode moving force analysis diagrams were for day 10 (B1) and for day 15 (B2). Nematodes that moved quickly away from the source of touch stimulus in a sinusoidal pattern were categorized as class a. Class b comprised nematodes that did respond to touch, but exhibited somewhat uncoordinated, slow movement. Nematodes unable to move forward or backward, but were clearly alive as seen by the slight movement of the head in response to touch, were classified as class c. The data was analyzed by one way-ANOVA analysis (SPSS 13) and different letters in a column denoted values that were significantly different (p < 0.05).

C. elegans display age-related deterioration in several physiologic processes, including a transition from rapid, coordinated body movement to sluggish, uncoordinated body movement; a decline in pharyngeal pumping rate; and increased intestinal lipofuscinosis and so on.³¹ The aforementioned parameters were analyzed to determine whether CP2-c2-s2 delays the age-related deterioration of physiologic processes. On day 10, all groups displayed rapid body movement. Most nematodes were in a spontaneous movement state (state a), and state c nematodes were not found (Fig. 5B1). On day 15, the number of nematodes in state b and c were significantly higher than in previous days (Fig. 5B2), and the number of state a nematodes in the 150 μg mL⁻¹ CP2-c2-s2 and 50 μg mL⁻¹ AcOH treatments was much higher than that of the control (P < 0.05), which indicates that CP2-c2-s2 significantly slowed down the decreased motility of C. elegans. The mean pharyngeal pumping rates decreased within 8 days (Fig. 5D). On day 4–8, the mean pharyngeal pumping rates decreased more slowly in the 150 μg mL⁻¹ CP2-c2-s2 and 50 μg mL⁻¹ AcOH treatments (P < 0.05) compared with that of the control, which indicates that CP2-c2-s2 delays the pharyngeal pumping rates of C. elegans to some extent. The Image Pro Plus software calculation revealed no significant differences in intestinal lipofuscin among the treatment and control groups on day 2 and day 6 (P > 0.05). However, the increase in lipofuscin in the 150 μg mL⁻¹ CP2-c2-s2 treatment was less than that of the control, which indicates that CP2-c2-s2 inhibits lipofuscin formation to some extent (data not shown). Nematodes are hermaphroditic worms, and their reproductive capacities were expressed in terms of the number of eggs laid. C. elegans deposited eggs mainly during the first 3 days in the treated and control groups (Fig. 5C) and no statistically significant differences were observed among these groups (P > 0.05), which indicate that CP2-c2-s2 does not affect fecundity.

C. elegans is a powerful model system for studying aging because of its well-established genetic pathways, relatively short lifespan, and the ease of propagation of synchronized individuals. These advantages have made C. elegans a popular model for studying aging and longevity that is becoming more popular than fruit flies and mice.³² Although C. militaris is widely used as a tonic or invigorant for longevity in China, its anti-aging activity has not been fully demonstrated. In this study, the anti-aging effects of CP2-c2-s2 from C. militaris were tested on C. elegans. The results demonstrate that CP2-c2-s2 significantly extends the mean and maximum lifespan by being able to slow down many age-related factors in the C. elegans model over time, such as reducing body movement, pharyngeal pumping rate, and lipofuscin accumulation. Moreover, locomotion ability declines on solid and liquid media with increasing age, which is correlated with the level of muscle deterioration.¹⁴ The decline of pharyngeal pumping, indicating the rhythmic contraction in the neuromuscular organ, is also one well-conserved feature of aging.³³ Lipofuscin is considered a hallmark of aging, and its accumulation is inversely correlated with longevity. Lipofuscin deposition ultimately decreases cellular adaptability until the cell finally undergoes apoptosis and it promotes the development of age-related pathologies.³⁴

The relationship between fecundity and longevity is studied by evolutionary biologists and developmental biologists using various model organisms. Tradeoffs, such as enhanced longevity at the expense of reduced fecundity, has been subject to intense research and speculation.³⁵ The tradeoff between fecundity and longevity is considered an important mechanism for survival during food shortages.³⁶ Some natural compounds extend the lifespan of C. elegans without reducing fecundity. Resveratrol extends the mean and maximum lifespan of C. elegans without causing a statistically significant decrease in fecundity.³⁷ Trehalose is a disaccharide of glucose found in diverse organisms that extends lifespan without causing losses in reproduction and other obvious side effects.³⁸ In this study, the results of the fecundity test were consistent with the aformentioned findings, and the beneficial effects of CP2-c2-s2 on lifespan were not associated with tradeoffs in terms of early life fitness in nematodes.

4. Conclusions

In conclusion, the polysaccharide CP2-c2-s2 was homogeneous, with an average molecular weight of 20 200 Da. CP2-c2-s2 is a β-pyran polysaccharide, probably with 1→2, 1→4, and 1→6 glycosyl linkages, and its structure needs to be studied further. The network structure of CP2-c2-s2 exhibited small aggregates, rod-like helical network structures, big ball-like aggregates, and large network structures from low to high concentrations. CP2-c2-s2 significantly stimulated proliferation of lymphocytes, (predominantly T- and B lymphocytes), extended the average lifespan without adverse effects on fecundity, and delayed age-related decreases in body movement, pharyngeal pumping rates, and lipofuscin accumulation in C. elegans. CP2-c2-s2 from C. militaris exhibited significant immunomodulatory and anti-aging activity. The potential of CP2-c2-s2 as an additive in functional foods and medicine should be explored further.

Acknowledgements

This research was partly supported by the Department of Education Research Project (Platform) of Guangdong Province (2013gjhz0003).

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