Antitumor activity of Dendrobium devonianum polysaccharides based on their immunomodulatory effects in S180 tumor-bearing mice

Abstract

The aim of the present study is to investigate the antitumor activity and immunostimulatory effect of the water-soluble polysaccharides (DDP) from stem of Dendrobium devonianum in S180 tumor-bearing mice. The mice were divided into 3 groups, and then DDP or Ganoderma lucidum polysaccharides (GLP) dissolved in deionized water were given to the mice at a dose of 200 mg kg⁻¹ for 30 days. DDP, as well as GLP, significantly inhibited the growth of mouse transplantable tumors and promoted spleen indexes, delayed-type hypersensitivity reaction, natural killer cell activity, macrophage phagocytosis and lymphocyte proliferation. Moreover, DDP and GLP increased the colonic acetate, butyrate and total short-chain fatty acid (SCFA) concentrations. These results indicated that oral administration of DDP significantly inhibited the growth of transplanted tumors in S180 tumor-bearing mice by improving specific and non-specific immune responses, as well as increasing colonic SCFAs.

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1. Introduction

It has been widely reported that medicinal plants such as Ganoderma lucidum could effectively inhibit the growth of tumor cells and improve immunity.^1–3 Like well-known Ganoderma lucidum, the antitumor and immune-enhancing activities of Dendrobium in mice were also reported.² Dendrobium belongs to Orchidaceae family, and it has been widely used in traditional Chinese medicine, tonic and diet in many oriental countries for a long time.^4,5 In addition to quercetin, an increasing amount of evidence has indicated that polysaccharides in Dendrobium can be considered as one of the most effective components exerting various pharmacological activities.⁶

Dendrobium devonianum Paxt. (D. devonianum) is primarily planted in Longling County, Baoshan City, Yunnan Province, China. It is also called purple leather Dendrobium in China for the purple skin of its mature stem. In contrast to other traditional species such as D. officinale, D. nobile, D. chrysotoxum and D. huoshanense, D. devonianum has higher yield and better quality.⁷ However, there are few reports on the bioactivity of D. devonianum, because it was just developed in China in recent years.

D. devonianum is a precious herbal plant highly valued in traditional Chinese medicine, and has been described in the Chinese Materia Medica Dictionary (2006 edition). The genetic relationship analysis of Dendrobium species by molecular marker systems in China showed that D. devonianum, D. officinale and D. nobile have close genetic kinship.⁸ In addition, it was discovered that D. officinale and D. nobile are deemed to be superior to other Dendrobium species according to the records of traditional medicine and modern medical evidence, and their bioactivities may be attributed to their polysaccharides.^4,5

Numerous studies have confirmed that polysaccharides are ideal immune modulators, which can not only improve host defense against pathogens, but also modulate adaptive immunity. This immunomodulatory activity was achieved by regulating the levels of lymphocytes, cytokines, chemokines, and antibodies.⁹ It has been reported that the water extract fraction, NaOH and HCl extract fractions were sequentially extracted from the stems of D. nobile to investigate the antitumor activities in Sarcoma 180 in vivo, and as a result, the water-soluble polysaccharide from D. nobile has better antitumor activity compared with alcohol- and salt-soluble polysaccharides.⁶ However, there is no information about the effect of water-soluble polysaccharides of D. devonianum on the growth of tumor cells and the immunity of mice.

In the present study, the water-soluble polysaccharide extract (DDP) was isolated from the stem of D. devonianum. DDP was orally administrated to S180 tumor-bearing mice to investigate the antitumor activity of the polysaccharides and the role of the immunostimulatory effect in cancer prevention. Ganoderma lucidum polysaccharides (GLP), some of the world-recognized anticancer and immune-enhancing polysaccharides, were used as positive control.

2. Methods and materials

2.1. Materials

Fresh D. devonianum was collected in Longling County, Baoshan City, Yunnan Province, China. The stems of D. devonianum were dried by freeze-drying and crushed into powder. GLP was purchased from Beijing Quanta Hengyi Technology Co. Ltd, Beijing, China.

The medium, penicillin, streptomycin and all other tissue culture reagents were purchased from GIBCO/BRL Life Technologies (Grand Island, NY, USA). Concanavalin A (ConA), lipopolysaccharide (LPS), Giemsa dye, 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethylsulfoxide (DMSO) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Chicken red blood cells (CRBCs) and sheep red blood cells (SRBCs) were purchased from Shanghai Rongbai Biological Technology Co. Ltd. Caco-2 cells were purchased from Cell Resource Center of Shanghai Institutes for Biological Sciences, CAS. Cell counting kit-8 (CCK-8) was purchased from Dojindo China Co. Ltd, Beijing, China. Gibco® RPMI Media 1640 was supplied by Life Technologies Corporation. LDH Cytotoxicity Assay Kit was purchased from Shanghai Beyotime Biotechnology Co. Ltd, Shanghai, China.

2.2. Preparation of water-soluble polysaccharides

After a reflux with methanol to remove impurities, the water-soluble polysaccharides were extracted three times from 200 g D. devonianum powder by 1.5 L deionized water at 80 °C for 4 h, and then centrifuged at 5000g at 4 °C for 10 min. The supernatant was concentrated to range from 1/4 to 1/5 volume of the original solution under reduced pressure, precipitated by ethanol (final concentration was 65%), and then centrifuged at 5000g at 4 °C for 10 min. The final precipitate was dissolved in distilled water, and then deproteinized with Sevag reagent until the protein absorption peak was not detected by UV.¹⁰ The solution was dialyzed with a MW_CO 7000 membrane against deionized water for 24 h at 4 °C while the water was changed every six hours. The supernatant was precipitated by ethanol as mentioned above and then freeze-dried. The obtained extractant was water-soluble polysaccharide extract (DDP).

2.3. Determination of polysaccharide purity

The polysaccharide contents of DDP and GLP were measured by the phenol–sulfuric acid method.¹¹ The purity of DDP and GLP was 90.7% and 91.2%, respectively.

2.4. Determination of average molecular weight

The molecular weight distributions and average molecular weights of DDP and GLP were determined according to our previous methods.¹² Briefly, the average molecular weights were measured by online multi-angle laser light scattering (DAWN EOS, Wyatt Technology Inc., USA) combined with gel permeation chromatography. The detector used a K5 cell and a He–Ne laser (λ = 690 nm), and differential refractive index detector (OPTILAB DSP). The column (TSK-gel G 4000 PWXL, TOSOH, Japan) was eluted with 0.1 M sodium nitrate solution at a flow rate of 0.5 mL min⁻¹. The concentration of each eluted fraction was determined by differential refractive index (ERC 7515 A) according to the known value of dN/dC = 0.15. The data were analyzed by Astra software (version 5.3.4, Wyatt).

2.5. Analysis of monosaccharide composition

For uronic acid quantification, DDP and GLP were measured after reduction by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). In brief, 10 mg of DDP and GLP were dissolved in 1 mL of distilled water, followed by an addition of 10 mg of EDC. This solution was incubated at room temperature for 1 h under constant pH 4.8 by HCI. Freshly prepared 2 M sodium borohydride (1 mL) was added to the reaction mixture twice during the next 1.5 h at 50 °C. The reaction was terminated by the addition of glacial acetic acid. The reaction mixture was dialyzed by a dialysis tube at 5000g. As for the monosaccharide composition of DDP and GLP, samples (10 mg) were hydrolyzed with 4 M trifluoroacetic acid solution at 80 °C for fructose and 120 °C for mannose, glucose, galactose, arabinose, xylose, and rhamnose for 2 h, followed by drying with nitrogen to remove trifluoroacetate after cooling to room temperature. The samples were diluted to 10 mL with distilled water. After filtration through 0.20 μm syringe filters, 10 μL samples were injected into the detector at 35 °C.

The monosaccharide compositions of DDP and GLP were determined by ion exchange chromatography (ICS-3000, DIONEX, USA) with a pulsed amperometric detector. The analytical column was a Carbo PacTMPA20 (3 × 150 mm, DIONEX, USA) with a Carbo PacTMPA20 BioLCTM guard column (3 × 30 mm). The mobile phase consisted of ultrapure water (solvent A), 250 mM sodium hydroxide solution (solvent B), and 1 M NaAC (solvent C). The flow rate was 0.5 mL min⁻¹. Gradient elution conditions were as follows. Condition 1: 0–20 min, 99% A, 1% B; 20–20.1 min, 94% A, 1% B, 5% C; 20.1–35 min, 79% A, 1% B, 20% C; 35–45 min, 20% A, 80% B; 45–55 min, 99% A, 1%B. Condition 2: 0–20 min, 94.0% A, 6.0% B; 20–20.1 min, 89.0% A, 6.0% B, 5.0% C; 20.1–35 min, 74% A, 6% B, 20% C; 35–45 min, 20% A, 80% B; 45–55 min, 94% A, 6% B. Monosaccharide content was quantified by response factors. Peaks were identified by comparing relative retention times with those of standards (99% purity, Sigma-Aldrich, Inc., USA).

2.6. NMR analysis

The sample for 1D and 2D NMR spectroscopy was prepared by dissolving 100 mg of DDP in 5 mL of D₂O and then freeze-drying; this dissolution-drying process was repeated twice. The sample recovered from the second freeze-drying treatment was re-dissolved in 4 mL of D₂O, passed through a nylon syringe filter (pore size 1.5 μm), and transferred into a regular NMR tube (5 cm). The ¹H NMR, ¹³C NMR, H–H cosy, H–C cosy and HMBC spectra of the sample were acquired, respectively, at 600 MHz and 150 MHz by a Bruker-600 NMR spectrometer. The chemical shifts were expressed in ppm using TMS as an internal standard.

2.7. Animals

Kunming mice (male, 25.0 ± 2.0 g, Permit no. SCXK (HU) 2012-0002) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China). The mice were housed in a stainless steel cage (22 ± 2 °C, 55 ± 5% humidity, and 12 hours light/dark cycle). Before the experiment, all the mice were acclimatized to the laboratory conditions for one week, and then assigned into 3 groups (control, DDP, and GLP, n = 10) so that the average body weights were the same for all groups. The mice had free access to standard laboratory diet (AIN-93G) while deionized water was allowed.

Mouse sarcoma S180 cells (Health Science Center of Beijing University) were maintained in vivo by intraperitoneal passage of 2 × 10⁶ cells in male Kunming mice for one week. The cell suspension was implanted subcutaneously to the fore right subaxillary of the mice. After inoculation, 1 mL samples were orally administered at 10:00 AM each day. DDP and GLP dissolved in deionized water were given to the mice at a dose of 200 mg kg⁻¹ body weight, and deionized water was orally administered to the control mice for 30 days. Mice were sacrificed 24 h after the final administration.

All experiments were carried out according to PR China legislation regarding the use and care of laboratory animals and were approved by the Bioethics Committee of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College.

2.8. Tumor inhibitory rate and organ indexes

After sacrificing by cervical dislocation on the 31^st day, mice were weighed, and their blood was collected by the retro-orbital plexus method, while their solid tumors, livers, spleens, and hearts were collected. The organ indexes were expressed as the organ weight relative to the body weight. The inhibition rate of tumor growth was calculated according to the following formula:

Tumor inhibitory rate (%) = [(A − B)/A] × 100

where A is the average tumor weight of the model control group; B is the average tumor weight of treated groups.

2.9. Antitumor test in vitro

Caco-2 and S180 cells were cultured in 48-well flat-bottom plates (2 × 10⁵ per well) for 24 h each. When the culture time was reached, 30 μL MTT solution (2 mg mL⁻¹, in PBS) was added to each well of the 48-well plates, and then medium containing 10, 50, 100, 200, 500 μg mL⁻¹ of DDP and GLP was introduced into the wells in triplicate. After incubation at 37 °C for 4 h, the medium containing MTT was removed, whereas 100 μL of cell dissociation solution (50% v/v DMSO, 20% w/v SDS) was added. The absorbance at 570 nm was measured using a Bio-Tek ELX808 (Bio-Tek Instruments, Inc. Winooski, VT, USA).

2.10. Delayed-type hypersensitivity reaction

All the mice were injected intraperitoneally with 0.2 mL 2% (v/v) sterile saline suspension of SRBCs on five days prior to the end of the feeding period. After that, the mice were injected intradermally in the left back sole with 20 μL of 20% SRBC solution on the day before the mice were sacrificed. The delayed-type hypersensitivity reaction (DTH) response was quantified by the extent of local inflammation determined by the increase in sole thickness 24 h after the second inoculation.

2.11. Assay of natural killer (NK) cell activity

Assay of NK cell activity was carried out according to a previous report.¹³ After the mice were sacrificed, their spleens were aseptically removed and placed into chilled RPMI-1640 medium. Erythrocytes were lysed with ammonium chloride buffer containing 0.01 M KHCO₃, 0.155 M NH₄Cl, and 0.1 mM EDTA. Lymphocytes were washed twice with RPMI-1640 and then cultured in 96-well plates at 5 × 10⁵ cells per well. The activity of NK cells was determined according to a previous report.¹⁴ K562 cells were added at 1 × 10⁴ cells/100 μL to make the effector/target ratio equal to 25 : 1. After the plates were incubated at 37 °C in 5% CO₂ atmosphere for 12 h, 20 μL solution containing 100 μg MTT was added to each well. After incubating for 4 h, 100 μL supernatant was removed from each well and discarded, whereas 100 μL cell dissociation solution (20% w/v SDS, 50% v/v DMSO, pH 4.5) was added, and then the plate was incubated at 37 °C for 12 h. The optical density value was measured by an automated plate reader (MQX200, Bio-Tek Instruments, Inc., Winooski, VT, USA) at 490 nm. NK cell activity was calculated by the following formula:

NK activity (%) = (T − (S − E))/T × 100%

where T is the optical density value of target cell control; S is the optical density value of test samples; E is the optical density value of effect cell control.

2.12. Macrophage phagocytosis

Macrophage phagocytosis of mice was measured according to a previous report.¹³ In brief, mice were sacrificed by cervical dislocation after their blood was collected by eyeball extirpation. In order to stimulate the mice, 4 mL Hank's solution was injected intraperitoneally, followed by quickly preparing peritoneal macrophages from peritoneal exudates. The macrophage phagocytosis on CRBCs was measured by adding Giemsa staining. The CRBC counts were observed by light microscopy (XSP-2CA, Shanghai Optical Instrument Co. Ltd, Shanghai, China). The phagocytic rate (PR) was calculated by the following formula:

PR (%) = (number of macrophage − ingesting CRBCs)/total number of macrophages × 100%.

2.13. Proliferation of splenic lymphocytes

Proliferation of splenic lymphocytes was measured by MTT incorporation according to a previous report.¹³ After isolating from spleen tissue by nylon mesh filters, the splenocytes were washed twice with RPMI-1640 and cultured in 96-well plates (5 × 10⁵ cells per well) with 10 μg mL⁻¹ LPS or 2 μg mL⁻¹ ConA. After incubation at 37 °C with 5% CO₂ atmosphere for 48 h, 20 μL solution containing 100 μg MTT was added to each well for another 4 h incubation. After centrifugation at 1400 × g for 5 min, the untransformed MTT was removed from each well and discarded, whereas 100 μL cell dissociation solution (20% w/v SDS, 50% v/v DMSO, pH 4.5) was added. The absorbance was measured by an automated plate reader (MQX200, Bio-Tek Instruments, Inc., Winooski, VT, USA) at 570 nm.

2.14. Short-chain fatty acids (SCFAs) analysis

Mice colon contents were collected and processed to detect SCFAs according to a previous report.¹⁵ In the chilled condition, 200 mg colon contents were homogenized in 500 μL distilled water, and acidified with 25% metaphosphoric acid at a ratio of 1 : 5 (v/v) for 30 min. After centrifuging at 12 000 × g at 4 °C for 10 min, the supernatant was collected for analysis. The SCFAs were measured by ion chromatography (DIONEX ICS-3000, USA) equipped with a high-performance capillary column (IonPac AS11-HC, 4 × 250 mm), a suppressor (ASRS 300 4 mm), and a guard column (IonPac AG11-HC, 4 × 50 mm). Gradient elution was performed by 0.8 mM KOH for 0–12 min, 0.8–34 mM KOH for 12–40 min, and 34 mM KOH for 40–50 min, while the column temperature remained at 30 °C. Acetate, propionate, butyrate and valerate (Sigma, USA) were used as standards.

2.15. Statistical analyses

The data were expressed as means with standard errors (SE) and analyzed by the Tukey–Kramer multiple comparison post hoc test. The analysis was carried out with SPSS (version 12.0 for Windows, SPSS Inc., Chicago, IL, USA). Differences were considered to be significant at P < 0.05.

3. Results

3.1. Molecular weight and monosaccharide composition of DDP

The yield of DDP was 10.7%, which was calculated against the wet weight of fresh D. devonianum. The molecular weight distributions of DDP and GLP were measured (Table 1 and ESI 1†). The average molecular weights of DDP and GLP were both more than 1 × 10⁵ g mol⁻¹. The monosaccharide composition of DDP, including mannose, glucose, galactose, galacturonic acid, fructose, glucuronic acid, arabinose, xylose, and rhamnose, was analyzed and the results are shown in Table 1. DDP had higher content of mannose (63.6%) and lower content of glucose (33.9%) compared with GLP.

Table 1 Molecular weight distribution and monosaccharide composition of DDP and GLP^a

	DDP	GLP
a Means were determined in triplicate for each cultivar.b Mass fraction. —, unidentified.
Molecular weight distribution
Mw (Da)
Peak 1	5.07 × 10^5 (12.0%)^b	1.20 × 10^6 (23.6%)
Peak 2	1.18 × 10^5 (85.9%)	2.87 × 10^4 (76.4%)
Peak 3	1.32 × 10^4 (2.1%)	—
Polydispersity (Mw/Mn)
Peak 1	1.15	1.48
Peak 2	1.59	2.42
Peak 3	1.05	—
Average molecular weight (Da)	1.22 × 10^5	3.04 × 10^5
Monosaccharide composition (%)
Mannose	63.6	1.33
Glucose	33.9	90.3
Galactose	0.84	1.83
Galacturonic acid	0.91	1.04
Fructose	0.24	2.37
Glucuronic acid	0.22	1.08
Arabinose	0.15	1.86
Xylose	0.04	0.17
Rhamnose	0.02	0.04

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3.2. NMR analysis

The NMR sample used for structural studies was viscous and showed relatively broad signals. Assignments of signals and identification of sugar residues were done by combinations of two-dimensional techniques and comparison of the chemical shifts with published data on similarly substituted sugar residues.^16–19 The ¹³C NMR chemical shift deviated about 2 ppm due to the difference of chemical shift standard. Based on the NMR spectra (Fig. 1), we speculated that the signals at 103.0 (Manp-C1), 72.8 (Manp-C2), 74.3 (Manp-C3), 79.4 (Manp-C4), 77.9 (Manp-C5) and 63.4 (Manp-C6) belonged to mannopyranose, and the signals at 4.83 (Manp-H1), 4.13 (Manp-H2), 3.83 (Manp-H3), 3.83 (Manp-H4), 3.57 (Manp-H5) and 3.93, 3.77 (Manp-H6) also belonged to mannopyranose. The chemical shift of 79.4 (Manp-C4) showed that the D-mannopyranose was 1→4 linked.

Fig. 1 ¹³C NMR, ¹H NMR and C–H cosy spectra of DDP in D₂O.

The peaks at 63.3 (Manp-C6) showed that the O-acetyl groups were at C-6 position of the mannopyranose. The signals at 188.6 ppm, 176.3 ppm, 175.8 ppm, 175.7 ppm, 23.4 ppm, and 23.2 ppm in the ¹³C NMR spectrum, together with the signals at 2.20 ppm, 2.18 ppm, 2.12 ppm and 1.92 ppm in the ¹H NMR spectrum, indicated that there were more O-acetyl groups linked to different carbon atoms besides the C-6 position of mannose residues. The ¹H NMR signal at 5.52 was attributed to H2 of 2,6-di-O-acetylmannopyranose and the ¹H NMR signal at 5.12 was attributed to H3 of 3,6-di-acetylmannopyranose. Therefore, the structure of DDP may be a mixture of 6-O-acetylmannopyranose, 2,6-di-O-acetylmannopyranose and 3,6-di-O-acetylmannopyranose.

3.3. Antitumor activities of DDP in vivo and in vitro

Growth and organ indexes of S180 tumor-bearing mice were determined and are shown in Table 2. The spleen weight and spleen index of S180 tumor-bearing mice in the DDP and GLP groups were significantly higher than those in the control group (P < 0.05). There was no significant difference in other indexes among the 3 groups.

Table 2 Effects of DDP on growth and organ indexes in S180 tumor-bearing mice^a

	Control	DDP	GLP
a Means expressed with SE were determined from 10 mice per group. Different superscript letters indicate significant differences at P < 0.05 (Tukey–Kramer multiple comparison post hoc test).
Food intake (g per day)	3.15 ± 0.21	3.17 ± 0.23	3.14 ± 0.19
Body weight (g)
Before treatment	32.5 ± 1.1	32.7 ± 0.9	32.6 ± 1.2
After treatment	44.5 ± 1.6	42.3 ± 2.1	43.5 ± 1.4
Body weight gain	12.0 ± 1.2	9.6 ± 1.8	10.9 ± 1.0
Organ weight
Spleen (mg)	98.3 ± 10.6^a	163.3 ± 11.8^b	167.5 ± 9.8^b
Liver (g)	1.97 ± 0.05	2.00 ± 0.09	1.95 ± 0.07
Heart (g)	0.22 ± 0.02	0.21 ± 0.02	0.20 ± 0.03
Organ index (mg g^−1)
Spleen	2.21 ± 0.64^a	3.86 ± 0.58^b	3.85 ± 0.49^b
Liver	44.3 ± 2.3	47.3 ± 3.4	44.8 ± 2.2
Heart	4.94 ± 0.31	4.96 ± 0.40	4.60 ± 0.34

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Tumor weight and tumor inhibitory rate in S180 tumor-bearing mice were analyzed and are shown in Fig. 2. The tumor weight of S180 tumor-bearing mice in the DDP and GLP groups was significantly lower than that of the control group (P < 0.05) (Fig. 2A). The tumor inhibitory rates in the DDP and GLP groups were 35.14% and 34.23%, respectively (Fig. 2B). However, in in vitro antitumor experiments, DDP and GLP did not have any effect on the growth of Caco-2 and S180 cells (ESI 2†).

Fig. 2 Tumor weight (A) and tumor inhibitory rate (B) of the experimental S180 tumor-bearing mice. Means and SE were determined from 10 mice per group. Different superscript letters indicate significant differences at P < 0.05.

3.4. Immune functions of DDP in S180 tumor-bearing mice

As shown in Fig. 3, the immunopotentiation of DDP was evaluated by DTH response, NK cell activity, and macrophage phagocytosis in S180 tumor-bearing mice. SRBC-induced DTH response, NK cell activity, and macrophage phagocytosis of CRBCs in the DDP and GLP groups were significantly enhanced in comparison to the control group (P < 0.05).

Fig. 3 DTH response (A), NK cell activity (B), and phagocytic rate (C) of the experimental S180 tumor-bearing mice. Means and SE were determined from 10 mice per group. Different superscript letters indicate significant differences at P < 0.05.

LPS-stimulated splenocyte proliferations in the DDP and GLP groups were significantly increased as compared with the control group (P < 0.05) (Fig. 4). In addition, the LPS-stimulated splenocyte proliferation in the DDP group was significantly higher than that in the GLP group (P < 0.05). ConA-stimulated splenocyte proliferation in the DDP group was significantly increased compared with the control and GLP groups (P < 0.05) (Fig. 4). There was no significant difference in ConA-stimulated splenocyte proliferation between the control and GLP groups.

Fig. 4 Splenocyte proliferations in the S180 tumor-bearing mice were stimulated by LPS and ConA. Means and SE were determined from 10 mice per group. White, control group; gray, DDP group; black, GLP group. Different superscript letters indicate significant differences at P < 0.05.

3.5. SCFAs content of colon contents

The acetate, butyrate, and total SCFA concentrations in the DDP and GLP groups were significantly higher than those in the control group (P < 0.05). Moreover, the three indexes in the DDP group were significantly higher than those of the GLP group (P < 0.05). The valerate in the DDP group was significantly higher than that in the control and GLP groups (P < 0.05). There was no significant difference in propionate concentration among the three groups.

4. Discussion

It has been extensively demonstrated that, as the main active ingredient of Ganoderma lucidum, GLP shows antitumor and immunomodulatory effects so that it has been widely used as an adjuvant of antitumor therapy in the clinic.^20,21 In the present study, we demonstrated that the oral administration of DDP at a dose of 200 mg kg⁻¹ body weight, as the same as GLP, significantly inhibited the growth of S180 tumors transplanted in mice compared with the model control (Fig. 2). In addition, no sign of toxicity was observed in the mice treated with DDP on the basis of body weight (Table 2). These results indicated that intake of DDP was one possible way to prevent the development of cancer.

This finding is consistent with a previous study reporting that the water-soluble polysaccharides from D. nobile could significantly inhibit growth of tumor cells in mice.⁶ This similar activity may be attributed to the close genetic kinship between D. devonianum and D. nobile.⁸ In general, the close genetic kinship is directly reflected in the monosaccharide compositions of the water-soluble polysaccharides from these two Dendrobium species. As shown in Table 1, similar to D. nobile polysaccharides, DDP has a high content of mannose which is positively correlated with anticancer activity.⁶ Furthermore, its superior water solubility as well as high molecular mass was considered to play an important role in its high antitumor activity. It is evident that glucomannan, a similar polysaccharide to DDP isolated from Dendrobium officinale, was composed of β-1,4-D-mannopyranosyl and β-1,4-D-glucopyranosyl residues, and glucomannan contained O-acetyl groups on Man residues.^16,17 Polysaccharides with a backbone of (1→4)-linked β-D-mannopyranosyl residues and β-D-glucopyranosyl residues showed good antitumor and immunostimulatory effects,²² which explained the relation between the structure of DDP from Dendrobium devonianum and its bioactivities.

Antitumor effects of many bioactive polysaccharides, such as those of Ganoderma lucidum, Dendrobium tosaense, and arabinoxylans, can be primarily attributed to their immune-enhancing activities.^23,24 Although the immunomodulatory activity of GLP is now well documented, little is known about the beneficial effects of DDP. As an important index of overall immune function, the DTH reaction is a cell-mediated pathologic response involved in T-cell activation and the productions of many cytokines.¹³ We demonstrated that DDP, as well as GLP, significantly enhanced spleen index (Table 2) and SRBC-induced DTH reaction (Fig. 3A) in tumor-bearing mice, which shows that DDP promotes overall immune function. NK cell activity assay is used to analyze a patient's cellular immune response in vitro and the antitumor activity of functional foods or drugs.^25,26 DDP has been confirmed to enhance the activity of inactivating NK cells from the splenocytes of tumor-bearing mice in the present study (Fig. 3B). This finding shows that DDP could enhance the cytotoxic activity against spontaneously derived tumor cells. In addition, DDP increased macrophage phagocytosis of CRBCs in the present study (Fig. 3C), which indicated its important role in macrophage activation.

Lymphocyte proliferative responses stimulated by LPS on B-cells and ConA on T-cells are often used to evaluate the functional capacity of T- and B-lymphocyte immunity.²⁷ In the present study, DDP significantly promoted the LPS- and ConA-stimulated splenocyte proliferation in tumor-bearing mice (Fig. 4). Moreover, the effect of DDP on the promotion of LPS-stimulated splenocyte proliferation was better than that of GLP (Fig. 4). These results showed that DDP could significantly enhance the humoral immunity and cell-mediated immunity by increasing the activation potential of T and B cells in tumor-bearing mice. In addition, these findings on the immunostimulatory activity of DDP are consistent with the results of reports about the water-soluble polysaccharides from the stems of D. huoshanense and D. tosaense.^2,28

Although immunomodulatory experiments confirmed that the antitumor activity of DDP was largely due to its immunostimulatory properties, whether itself or its metabolites are responsible for the effects is still unknown. Our results showed that treatment with DDP could not inhibit the growth of Caco-2 and S180 cells in vitro (data not shown). This result indicated that the antitumor effect mechanism of DDP is possibly related to its metabolites in vivo. As for many bioactive polysaccharides, like prebiotics, their end products fermented in vivo contain more SCFAs.^24,29 Ohtani³⁰ reviewed numerous articles about the relationship of microbiome and cancer, and summarized that the SCFAs, acetate, propionate, and butyrate, function in the suppression of inflammation and cancer. In addition, these SCFAs are able to improve mucosal morphology by increasing mucin production and decreasing translocation binding with SCFA receptors to immune cells, thereby enhancing the immunity of the body.^31–33 In the present study, acetate, butyrate and total SCFA concentrations in the DDP and GLP groups were higher than those in the control group (Table 3). This indicated that the antitumor effects and immunomodulatory activities of DDP might be related to its intestinal prebiotic functions.

Table 3 Effects of DDP on SCFA concentrations in S180 tumor-bearing mice^a

	Control	DDP	GLP
a Means expressed with SE were determined from 10 mice per group.b The data of fecal SCFAs are the sum of acetate, propionate, butyrate, and valerate. Different superscript letters indicate significant differences at P < 0.05 (Tukey–Kramer multiple comparison post hoc test). SCFAs: short-chain fatty acids.
SCFAs (μmol g^−1)
Acetate	26.32 ± 1.83^a	43.63 ± 2.93^c	35.13 ± 2.38^b
Propionate	5.51 ± 0.61	7.50 ± 1.12	7.35 ± 1.13
Butyrate	3.09 ± 0.43^a	5.93 ± 0.49^c	4.87 ± 0.58^b
Valerate	0.19 ± 0.05^a	0.61 ± 0.06^b	0.24 ± 0.05^a
Total SCFAs^b	35.10 ± 2.15^a	57.67 ± 3.01^c	47.59 ± 2.89^b

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Moreover, the above three indexes in the DDP group were significantly stronger than those in the GLP group (P < 0.05). This may be one of the reasons why DDP showed stronger capacity of promoting LPS-stimulated splenocyte proliferation than GLP. As is well known, the type and quantity of bioactive polysaccharide fermentation end-products depend on their monosaccharide composition and structure.^29,34 The difference in physicochemical parameters between DDP and GLP might induce the disparity in colonic SCFA concentrations between the two groups. However, the specific structure–activity relationship should be further studied.

5. Conclusions

The present study clearly showed that oral administration of DDP significantly inhibited the growth of transplanted tumors by improving specific and non-specific immune responses such as DTH response, NK cell activity, macrophage phagocytosis and lymphocyte proliferation, as well as increasing colonic SCFAs in S180 tumor-bearing mice. DDP may be developed as a novel antitumor agent with immunomodulatory activity.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant no. 31401505) and Core Research Budget of the Non-profit Governmental Research Institution (ICS, CAAS) (grant no. 1610092015002-03).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra03074b

‡ Equal contributors.

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