Botanical Studies (2007) 48: 63-70.

*

Corresponding author: E-mail: wchou@tmu.edu.tw; Phone:

+886-2-2736-1661 ext. 6160; Fax: +886-2-2378-0134.

Introduction

Yam ( Dioscorea species) is a member of the mono-

cotyledonous family Dioscoreaceae and is a staple food in

West Africa, Southeast Asia, and the Caribbean (Akoruda,

1984). The fresh tuber slices are widely used as functional

foods in Taiwan, and the dried slices are used as traditional

Chinese medicines (Liu et al., 1995). Yam tuber contains

mucilages, mannan-protein macromolecules (Misaki et al.,

1972; Tsai and Tsai, 1984). Recently, yam tuber mucilage

was reported to exhibit antioxidant (Hou et al., 2002; Lin

et al., 2005), angiotensin converting enzyme inhibitory

activities (Lee et al., 2003) and hypoglycemic activities

(Hikino et al., 1986; Bailey and Day, 1989). Furthermore,

Chinese yam (D. alata cv. Tainong No. 2) feeding re-

sulted in antioxidant effects in hyperhomocysteinemia rats

(Chang et al., 2004).

Many isolated polysaccharides are reported to have

immunomodulatory activities (Brown and Gordon, 2003;

Feizi, 2000), and medicinal mushrooms (Wasser, 2002)

have been intensively investigated for their beneficial ef-

fects as immunomodulatory and antitumor agents. Len-

tinan (Len), the (1¡÷3)-

£]

-glucan isolated from Lentinus

edodes, has been demonstrated to have an anti-tumor

activity against Sarcoma 180 in vivo and in vitro (Zhang

et al., 2005). Reishi (Ganoderma lucidum) polysaccha-

rides were reported as immune potentiators (Chang and

Lu, 2004; Zhu and Lin, 2005; Hsu, et al., 2004). The cold-

water extracts of dietary mushrooms, including Hypsizigus

mamoreus, Agrocybe aegerita, and Flammulina velutipes,

were showed to have antiproliferative activity against

human leukemic U937 cells (Ou et al., 2005). The im-

munomodulatory activity by an isolated

£\

-glucan-protein

complex from mycelium of Tricholoma matsutake has also

been documented (Hoshi et al., 2005). Several food-grade

microalgae, including Spirulina platensis, Aphanizomenon

flos-aquae, and Chlorella pyrenoidosa, are also known to

contain polysaccharides, potent immunostimulators of hu-

man monocytes and macrophages (Pugh et al., 2001). In

this study, orally administered mucilages from three dif-

ferent Taiwanese yam cultivars, including Dioscorea alata

L. cv. Tainong 1 (TN1), Dioscorea alata L. cv. Tainong 2

Immunostimulatory activities of yam tuber mucilages

Huey-Fang SHANG

1

, Huey-Chuan CHENG

2

, Hong-Jen LIANG

3

, Hao-Yu LIU

4

, Sin-Yie LIU

5

, and

Wen-Chi HOU

4,

*

1

Department of Microbiology and Immunology, Taipei Medical University, Taipei, Taiwan

2

Mackay Memorial Hospital, Taipei 104 and Mackay Medicine, Nursing and Management College, Taipei 112, Taiwan

3

Department of Food Science, Yaunpei University of Science and Technology, Hsinchu 300, Taiwan

4

Graduate Institute of Pharmacognosy, Taipei Medical University, No. 250, Wu-Hsing Street, Taipei 110, Taiwan

5

Taiwan Agricultural Research Institute, Council of Agriculture, Executive Yuan, Wu-Feng, Taichung, Taiwan

(Received May 9, 2006; Accepted August 15, 2006)

ABSTRACT.

The purified mucilages from three Taiwanese yam cultivars, including Dioscorea alata L. cv.

Tainong 1 (TN1), D. alata L. cv. Tainong 2 (TN2), and D. alata L. var. purpurea (Roxb.) cv. Ming-Jen (MJ),

and the commercial lentinan (Len) were used to evaluate the immunostimulatory effects on the murine innate

and adaptive immunity. BALB/c mice were grouped and administrated orally with 0.5 ml of TN1, TN2, MJ

daily for 5 weeks. The positive and negative controls were fed with lentinan and distilled water, respectively.

Blood samples were drawn from the retroorbital sinus on day 7 and 21, and the lymphocyte subpopulation,

phagocytosis of granulocyte and monocyte were analyzed by flow cytometry. The mice were sacrificed on

day 36, and the splenocytes were harvested for determinations of natural killer (NK) cell cytotoxicity activ-

ity. The stimulation index on the phagocytosis of peritoneal macrophages and the RAW264.7 cell line by yam

mucilage were also determined in vitro. The results showed that all three mucilages, especially MJ yam, could

elevate the number of T helper cells in the peripheral blood and enhance the phagocytic activity of granulo-

cyte, monocytes and macrophages both ex vivo and in vitro tests. Increased splenic cytotoxic activity follow-

ing the administration of mucilages from MJ yam was observed. Furthermore, the production of specific anti-

ovalbumin (OVA) antibody and OVA-stimulated splenic cell proliferation were also enhanced by all mucilage

groups. It is suggested that the tuber mucilage may function as an immunomodulatory substance.

Keywords: Dioscorea; Immunostimulatory; Lentinan; mucilage; Nk cell; Phagocytic activity; Yam.

BIOCHEMISTRY

64

Botanical Studies, Vol. 48, 2007

(TN2), and D. alata L. var. purpurea (Roxb.) cv. Ming-Jen

(MJ), were evaluated for their immunostimulatory effects

on the innate and adaptive immunity of BALB/c mice.

MATERIAL AND METHODS

Plant materials

Local fresh tubers of Taiwanese yam, including D. ala-

ta L. cv. Tainong 1 (TN1), D. alata L. cv. Tainong 2 (TN2),

and D. alata L. var. purpurea (Roxb.) cv. Ming-Jen (MJ),

were kindly provided by Dr. Liu, Sin-Yie (Taiwan Agricul-

tural Research Institute, Wu-Feng, Taichung, Taiwan). The

TN1 tuber is a round or elliptic shape with white fleshes

in the brown peel. The TN2 tuber is a cylindraceous shape

with white fleshes in the brown peel. The MJ tuber is a

cylindraceous shape with purple fleshes in the purplish-red

peel.

Extraction and purification of yam tuber

mucilage

After washing and peeling, the yam tubers were cut into

strips for mucilage extraction and purification as described

elsewhere (Hou et al., 2002; Lee et al., 2003). Briefly, yam

tuber was homogenized with four volumes (W/V) of 50

mM Tris-HCl buffer (pH 8.3) containing 1% vitamin C.

After centrifugation at 14,000

¡Ñ

g for 30 min, the superna-

tants were mixed with isopropanol to a final concentration

of 70%, and stirred quickly at 4oC overnight. The precipi-

tates were filtrated and dehydrated with 100% isopropanol,

then, rinsed with acetone. After drying at 40oC in an oven,

the crude mucilage (CM) was ground and collected for

further purification by both SDS and heating procedures.

One gram of CM powder was dissolved in 200 ml distilled

water and kept in a 50oC water bath. Forty mililiters of 5%

SDS solution (dissolved in 45% ethanol) were added to

the CM solution. The mixture was stirred gently at 50oC

for 30 min, then, at room temperature for another 2 h.

The mucilage solution was then placed in an ice bath to

quickly reduce the temperature in order to precipitate the

SDS-protein complex. After centrifugation as above, the

supernatants were precipitated with isopropanol and dried

in a 40oC oven as described earlier. The mucilage was

again ground, dissolved, and then heated in boiling water

for 20 min. After centrifugation, the supernatants were

mixed with isopropanol to a final concentration of 70%.

The partially purified mucilage was filtrated, dehydrated,

rinsed with acetone, dried, and then collected for further

use.

Experimental animals

Five weeks old male BALB/c mice were purchased

from National Laboratory Animal Center (Taipei, Taiwan)

and divided randomly into five groups (n=8). Each group

was housed individually in wire-bottomed stainless steel

cages in a temperature- and humidity-controlled room

(at 22oC) with a 12-h light/dark cycle and free access to

AIN-76 feeds and water. All animal experimental proce-

dures followed the published guidelines (COA, 2004). For

assessment of innate immunity, each 0.5 ml of TN1, TN2,

MJ (10 mg/m) and commercial Len (0.05 mg/ml) were ad-

ministrated orally once a day for 5 weeks. Distilled water

was used for the control group. Blood samples were drawn

from the retroorbital sinus on days 7 and 21, and the lym-

phocyte subpopulation and both phagocytosis of granulo-

cyte, and monocyte were analyzed by flow cytometry as

described below.

Lymphocyte subpopulation assay

The labeled primary antibodies used for lymphocyte

subpopulation, including T cell, B cell, T helper cell, and

cytotoxic T cell, by flow cytometry (Viau et al., 2005)

were showed in Table 1. All labeled monoclonal antibod-

ies were purchased from Serotec Company (Oxford, UK).

Fifty microliters of blood isolated from the retroorbital

sinus on days 7 and 21 were put in the Falcon tube (Falcon

2052). Labeled monoclonal antibodies (5 £gl) were added

and incubated at room temperature under light protec-

tion for 20 min. After lysis and washing, the lymphocyte

subpopulation was determined by the flow cytometry and

analyzed by CellQuest software (Becton Dickinson FACS

Calibur

TM

, CA).

Phagocytosis of granulocyte and monocyte

FITC-labeled Escherichia coli (Molecular Probes,

USA) powder (5 mg) was suspended in 0.5 ml of Hank¡¦s

balanced salt solution (HBSS) and used for phagocytosis

analysis by flow cytometry (Butcher et al., 2001; Gaforio

et al., 2002). One hundred microliters of blood from the

retroorbital sinus on days 7 and 21 were mixed with 20

£gl of FITC-labeled E. coli solution at 37oC for 10 min.

The Falcon tube was immersed in an ice bath to stop the

phagocytosis. One hundred microliters of of trypan blue

(1.25 mg/ml) were added to quench the residual FITC-

labeled E. coli. After lysis and washing, 5 £gl of propidium

iodide (PI, 2 mg/ml) were added for 10 min, and the

phagocytosis of granulocyte and monocyte was deter-

mined by flow cytometry (Becton Dickinson FACS Cali-

bur

TM

, CA).

Table 1. The labeled primary antibody used for lymphocyte subpopulation assay by flow cytometry.

Lymphocyte subpopulation

Surface marker Monoclonal antibody used

T cell

CD3

+

Phycoerythrin (PE)-anti-mouse CD3

B cell

CD19

+

Fluorescein isocyanate (FITC)-anti-mouse CD19

T helper cell

CD4

+

FITC-anti-mouse CD4

T cytotic cell

CD8

+

PE-anti-mouse CD8

SHANG et al. ¡X Immunostimulatory activities of yam tuber mucilages

65

Phagocytosis of peritoneal macrophage and

the RAW264.7 cell line in vitro

The peritoneal macrophages were obtained at 3 day

after intraperitoneal injectionior with 2 ml of thioglycol-

late broth (Becton Dickinson, CA) (Choi and Hwang,

2002; Choi et al., 2004). RAW264.7 cell line (Sosroseno

et al., 2003) was cultured in Dulbecco¡¦s modified eagle

medium (DMEM, GibcoBRL, USA) containing 5% fetal

bovine serum (FCS, GibcoBRL, USA), adjusted to 2

¡Ñ

10

6

cell/ml, and seeded in a 96-well plate (100

£g

l/well). Ten

microliters of TN1, TN2, MJ (1 mg/ml), and commercial

Len (0.1

£g

g/ml) were added to the 96 wells and cultured

in a 5 % CO

2

humidified incubator at 37oC for 60 min.

After removing the supernatants, 20

£g

l of FITC-labeled E.

coli was added for 2 h. Two hundred microliters of trypan

blue (1.25 mg/ml) were added in each wells one min for

quenching. The stimulation index (%) for phagocytosis of

peritoneal macrophage and RAW264.7 cells by yam muci-

lages was determined by fluorescent analyzer.

Splenocyte-mediated cytotoxicity assay

Treated mice were sacrificed at day 36 and splenocytes

were harvested for determinations of NK cell activity

(Choi et al., 2004; Zhu and Lin, 2005). Briefly, isolated

splenocytes were washed with PBS for 3 times and adjust-

ed to 4

¡Ñ

10

6

cell/ml with RPMI-1640 medium (GibcoBRL,

USA) as effector cells. The YAC-1 cells, as target cells,

were washed with HBSS and adjusted to 2

¡Ñ

10

6

cell/ml.

Target cells at 200 £gl were mixed with 2 £gl of DiOC

18

(3

mM) (Molecular Probes, USA) at 37oC for 20 min. Target

cells were resuspended in 200

£g

l of RPMI-1640 medium

(GibcoBRL, USA) for further uses. The effector cells were

mixed with target cells (40:1, 20:1, 10:1, and 5:1) in se-

rial dilutions and were co-cultured in 5% CO

2

humidified

incubator at 37oC for 2 h. The supernatants were removed,

and the same volume of PI solution (0.2 mg/ml) was add-

ed. The splenocyte-mediated cytotoxicity was determined

by flow cytometry (Becton Dickinson FACS Calibur

TM

,

CA).

Effects of yam mucilage on the production

of the specific anti-Ova antibody (adaptive

immunity assessments)

Mice were divided in 5 groups, each consisting of 8

animals and administered orally with 0.5 ml of TN1, TN2,

MJ (10 mg/ml) and commercial Len (0.05 mg/ml) once

a day for 5 weeks. The ovalbumin (OVA, 2 mg/ml was

used on day 0, and 6 mg/ml was used on day 21) was pre-

mixed with an equal volume of aluminum adjuvant (2.5

mg/ml) for mice immunizations. Each mouse was injected

intraperitoneally on day 0 and day 21 with 0.2 ml of OVA/

aluminum adjuvant. Blood samples were drawn from the

retroorbital sinus before and on days 7, 32, and 43 after

immunization. The production of specific anti-OVA an-

tibody was analyzed by ELISA method (Ml.kova et al.,

2001; Okamoto et al., 2003). The 96-well plate was coated

with OVA (1 to 5

£g

g/ml) and then blocked with bovine

serum albumin (10 mg/ml). Peroxidase-conjugated goat

anti-mouse IgG (diluted in 1:10

4

) or -conjugated goat anti-

mouse IgM (diluted in 1:10

4

) was added and incubayed at

room temperature for 2 h. The amounts of anti-OVA-IgG

and anti-OVA-IgM were measured by adding hydrogen

peroxide and TMB for 15 min and then recording at 450

nm by ELISA reader (TECAN Sunrise microplate reader,

Mannedorf, Switzerland).

Effects of yam mucilage on the splenocyte

proliferations after specific Ag stimulation

(adaptive immunity assessments)

Twenty-four days after the second booster, the mice

were sacrificed, and the splenocytes were harvested for

proliferation determinations by MTT assay. Briefly, iso-

lated splenocytes were washed with PBS for 3 times,

adjusted to 1

¡Ñ

10

6

cell/ml with RPMI-1640 medium

(GibcoBRL, USA), and seeded in a 96-well plate (100

£g

l/well). Fifty microliters of OVA

¡]

1 £gg/ml

¡^

were added

to each test well and then cultured in a 5% CO

2

humidi-

fied incubator at 37oC for 48 h. Five microliters of MTT

(5 mg/ml) were then added under light protection for 4

hrs and 100 £gl of 10% SDS in 0.01 N HCl were added for

18 hrs. Absorbance at 595 nm was determined by ELISA

reader (TECAN Sunrise microplate reader, Mannedorf,

Switzerland). The stimulation index (%) for proliferation

of splenocytes by OVA was calculated following the equa-

tion: (A

595

with yam mucilage treatment)

¡Ò

(A

595

without

yam mucilage treatment)

¡Ñ

100%.

Statistical analysis

Means of triplicates were measured. Student¡¦s t-test was

used for comparison between two treatments. A difference

between the control and each treatment was considered

statistically significant when P<0.05 (*) or P<0.01 (**).

RESULTS AND DISCUSSION

Yam tuber contains mucilages that were reported to be

a mannan-protein macromolecule (Misaki et al., 1972;

Tsai and Tai, 1984). Our previous report revealed that the

total recovery of purified yam tuber mucilages was about

48% that of the crude mucilages (Hou et al., 2002). In this

report, mucilages from three cultivars of native Taiwanese

yam were reported to have immunomodulatory activities

in innate immunity and adaptive immunity.

Lymphocyte subpopulation assay

The results showed that the number of T cells in TN2

or MJ yam mucilages-treated animals is increased as com-

pared with the control group in the first week (Figure 1A)

and MJ yam mucilages-treated ones in the third week (Fig-

ure 1B) (P<0.01). This cell increment was due to elevated

number of T helper cells and T cytotoxic cells (Figures 1C

and 1D) (P<0.05). The increased T helper cells were also

found after glutamine supplements in rats with gut-derived

sepsis (Yeh et al., 2004). Sinclair (1998) found that the

66

Botanical Studies, Vol. 48, 2007

the third, but not the first week as compared with the con -

trol (P<0.01). In the third week (Figure 2B), the phago-

cytic activities of granulocyte and monocyte populations

significantly increased in the Len (P<0.01), TN1 (P<0.05),

TN2 (P<0.05), and MJ (P<0.01) groups. Our present data

reveal that the oral administration of native Taiwanese

yam mucilages can elevate the phagocytic cell populations

of BALB/c mice ex vivo. In mammals, phagocytosis is a

very important defense against pathogen invasions and

apoptotic cell scavenging, which is performed by phago-

cytes like macrophages, dendritic cells, and granulocytes

(Stuart et al., 2005). Monocytes and other leukocytes are

recruited to the inflammatory site and differentiate in ad -

vance to inflammatory macrophages (Van den Berg et al.,

2001).

isolated polysaccharides from Astragalus membranaceus

(Huang-gi, Chinese herb) elevated the T cell and T cyto-

toxic cell numbers. The increased T helper cell subpopula-

tions in Th1 or Th2 subsets (Mosmann and Sad, 1996) and

the secreted cytokines will be further investigated.

Phagocytosis of granulocyte and monocyte

isolated from blood of BALB/c mice

In the first week (Figure 2A), the phagocytic activity of

the granulocyte population showed no apparent differenc-

es from the control group. However, phagocytic activity

by monocyte population significantly increased in TN1,

TN2, and the MJ groups as compared with that in the con-

trol (P<0.05 for TN1 And TN2 and P<0.01 for MJ). The

phagocytic activity in Len-treated animals was elevated in

Figure 1. The effects of yam mucilages on the lymphocyte subpopulations. The blood from the retroorbital sinus on day 7 and day 21

was analyzed for both T and B cells (A and B) and T cell subsets (C and D) using flow cytometry. Means of triplicates were measured.

A difference between the control and each treatment was considered statistically significant when P<0.05 (*) or P<0.01 (**). Bar rep-

resents standard deviation.

Figure 2. The effects of yam mucilages on the phagocytosis of granulocyte and monocyte on day 7 (A) and day 21 (B). Means of trip-

licates were measured. A difference between the control and each treatment was considered statistically significant when P<0.05 (*) or

P<0.01 (**). Bar represents standard deviation.

SHANG et al. ¡X Immunostimulatory activities of yam tuber mucilages

67

Phagocytosis of peritoneal macrophage and

the RAW264.7 cell line

The peritoneal macrophages and the RAW264.7 cells

were frequently used for phagocytic analysis in vitro (Choi

et al., 2004; Sosroseno et al., 2003). All the yam muci-

lages enhanced phagocytosis by murine peritoneal macro-

phages as compared with the control (P<0.01 for TN1 and

P<0.05 for TN2 and MJ, Figure 3). Similarly, all the yam

mucilages augmented phagocytosis by RAW264.7 cells

as compared with the control (P<0.01, Figure 3). Choi et

al. (2004) reported on the yam mucilages with stimulated

activity of phagocytosis. Our present result showed that

the stimulation index of peritoneal macrophage treated

with TN1, TN2, MJ, and commercial Len were 2.62, 1.34,

1.24, and 3.48-folds, respectively (Figure 3A), and the

stimulation index of RAW264.7 cell line treated with TN1,

TN2, MJ, and commercial Len were 2.73, 3.28, 3.17, and

6.7-folds, respectively (Figure 3B). High mannuronic acid-

containing alginate was reported to stimulate the murine

peritoneal macrophage phagocytosis (Son et al., 2001).

From the results of Figures 2 and 3, the mucilages from

three Taiwanese yams may exhibit a stimulatory effect

on phagocytic activity by granulocyte and monocyte (ex

vivo), on peritoneal macrophages, and on the RAW264.7

cells (in vitro).

Figure 3. The effects of yam mucilages on the phagocytic activity of peritoneal macrophage (A) and the RAW264.7 cell line (B).

Means of triplicates were measured. A difference between the control and each treatment was considered statistically significant when

P<0.05 (*) or P<0.01 (**). Bar represents standard deviation.

Figure 4. The effects of yam mucilages on the splenocyte-me-

diated cytotoxicity. Means of triplicates were measured. A dif-

ference between the control and each treatment was considered

statistically significant when P<0.05 (*) or P<0.01 (**). Bar

represents standard deviation.

Figure 5. Effects of yam mucilages on the production of the serum specific anti-ovalbumin antibody of IgM (A) or IgG (B). Means of

triplicates were measured. A difference between the control and each treatment was considered statistically significant when P<0.05 (*)

or P<0.01 (**). Bar represents standard deviation.

68

Botanical Studies, Vol. 48, 2007

Splenocyte-mediated cytotoxicity assay

The results of the present study showed that oral admin-

istration of mucilages promoted NK cell activity. The MJ

mucilages showed significant differences (40:1, P<0.01;

20:1, 10:1, 5:1, P<0.05) from the control group. Several

reports have shown that polysaccharides can activate NK

cell activity (Wasser, 2002; Choi et al., 2004; Vetvicka and

Yvin, 2004; Hoshi et al., 2005; Zhu and Lin, 2005). NK

cells are large granular lymphocytes that lyse a variety of

transformed and infected cells and are sufficiently devel-

oped to control infection or tumor (Diefenbach and Raulet,

2001). The potential use of MJ mucilages for anti-cancer

treatment will be further investigated.

Effects of yam mucilage on the production of

the specific anti-Ova antibody

For evaluating the effects of mucilages on the adap-

tive immunity, the production of specific IgM (Figure

5A) or IgG (Figure 5B) against OVA was determined.

It was found that one week after immunization the oral

administrations of mucilage could elevate the amounts

of specific IgM (Figure 5A) and specific IgG (Figure 5B)

against OVA compared to the control group (P<0.01, for

TN1 and TN2). After the second booster of OVA (day 21),

the specific IgM production (day 32 and day 43) remained

constant, and no significant differences among groups ap-

peared. However, the IgG production apparently increased

at day 32, and the Len and TN1 groups significantly out-

perfomed the control group (P<0.01). It was found that

the specific antibodies rapidly responses against foreign

proteins (or antigens) in the presence of yam mucilages.

It might be possible to use the yam mucilages as immune

adjuvants for immunomodulations.

Effects of yam mucilage on the splenocyte

proliferations

Following immunization with OVA, mice were sacri-

ficed on day 45, and the splenocytes were harvested for

proliferation determinations. As seen in Figure 6, out of

yam mucilages investigated, TN1 and MJ significantly

increased cell proliferation as compared with the control

(P<0.05). It meant that the yam mucilages might be acted

as mitogens. The increased lymphocyte subpopulation

needed investigations further.

In conclusion, the effects on the innate immunity (Fig-

ures 1 to 4) and adaptive immunity (Figures 5 and 6) of

oral administrations of three mucilages from Taiwanese

yams on BALB/c mice seem significant. It is suggested

that the tuber mucilage might function as an immunostim-

ulatory substance. The production of cytokines and anti-

cancer treatments will be investigated in the future.

Acknowledgements. The authors want to thank the Na-

tional Science Council (NSC 94-2313-B-038-001, NSC

94-2313-B-038-004), Republic of China for financial sup-

ports.

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Botanical Studies, Vol. 48, 2007