Immunopotentiating Activity of Dendrobium Species in Mouse Splenocytes

doi:10.4236/cm.2011.23017

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Chinese Medicine, 2011, 2, 103-108

doi:10.4236/cm.2011.23017 Published Online September 2011 (http://www.SciRP.org/journal/cm)

Immunopotentiating Activity of Dendrobium Species in

Mouse Splenocytes

David T. W. Lau, Michel K. T. Poon, Hoi Yan Leung, Kam Ming Ko

Division of Life Science, Hong Kong University of Science & Technology, Hong Kong, China

E-mail: [email protected]

Received April 4, 2011; revised April 25, 2011; accepted May 13, 2011

Abstract

This study aimed to explore a pharmacological activity marker for quality assurance of Dendrobium species.

The immunopotentiating activity in aqueous extracts prepared from four Dendrobium species, including D.

officinalis, was assessed by an in vitro assay of concanavalin A (Con A)-stimulated proliferation of mouse

splenocytes. Four samples of commercially available Dendrobii Caulis were also analyzed for comparison.

The results indicated that the aqueous extract of D. officinalis produced immunopotentiating action, as evi-

denced by the increase in Con A-stimulated proliferation of mouse splenocytes, with the extent of stimula-

tion being more prominent than those of other tested Dendrobium species and Dendrobii Caulis samples. In

conclusion, an in vitro immunopotentiation assay may be used for assessing the pharmacological activity of

Dendrobium species. The finding that D. officinalis produced a more potent immunopotentiating action is

consistent with its “yin-nourishing” action in Chinese medicine, which is more effective than other Den-

drobium species in clinical use.

Keywords: Dendrobium offincalis, Dendrobii Caulis, Splenocyte Proliferation, Concanavalin A

1. Introduction

Dendrobium species was regarded as a first-rate herb in

Shen-nong Bencao Jing (Cano n on Medicinal Herbs, 200

B.C.) and has a long history of usage in China. Over the

past few decades, the dried stem of Dendrobium species

(Dendrobii Caulis, also called Shi Hu in Chinese) has

been used for clearing “heat” and nourishing “yin” in the

practice of Chinese medicine [1]. A recent study showed

that the administration of an aqueous extract of Den-

drobium candidum ameliorated the dry-mouth symptom

in patients suffering from Sjögren syndrome [2]. Phar-

macological studies have demonstrated that the aqueous

extract and/or polysaccharides of Dendrobii Caulis pos-

sess antioxidant [3,4], immuno-stimulating [5-7], anti-

tumor [8,9], anti-microbial [9], anti-hyperlipidemia [10]

and anti-hyperglycemic [11] activities. The widespread

application of Dendrobii Caulis has raised a concern re-

garding the identity of Dendrobium species as herbal

source. In this regard, the record of Dendrobii Caulis in

Chinese Pharmacopoeia has been constantly amended

over the past 20 years. While five Dendrobium species,

including D. loddigesii Rolfe, D. fimbriatum Hook and D.

nobile Lindl., were listed in the 1995 and 2000 editions,

a more extended list of Dendrobium species was gener-

ated in the 2005 edition. As such, up to 74 Dendrobium

species can be used as Dendrobii Caulis in China. In the

2010 edition of Chinese Pharmacopoeia [12], D. Offici-

nalis has been singled out as a distinct herb originated

from D. officinale Kimura et Migo. Not surprisingly, D.

Officinalis is relatively scarce in abundance and deemed

to possess a stronger therapeutic potential than other

Dendrobium species [13,14].

As more than 45 Dendrobium species have been iden-

tified to possess therapeutic potential [15-18], the devel-

opment of chemical and DNA fingerprinting methods for

species authentication, particularly for those recorded in

Chinese Pharmacopoeia, has been an area of intensive

research [19-26]. In addition to chemical markers, the

measurement of pharmacological activity may offer a

complementary and functionally relevant quality assess-

ment for herbal materials [27]. In this connection, “yin-

nourishing” tonic herbs (including Herba Dendrobii)

have been found to produce immunopotentiating effect

on mouse splenocytes both in vivo and in vitro [28]. In

the present study, using an in vitro assay of conc anavalin

A (Con A)-stimulated proliferation of mouse splenocytes,

we assessed the immunopotentiating activity of aqueous

D. T. W. LAU ET AL.

104

extracts prepared from four Dendrobium species includ-

ing D. Officinalis, with an objective of exploring a pha-

rmacological activity marker for quality assurance. Four

samples of commercially available Dendrobii Caulis

were also analyzed for comparison.

2. Materials and Methods

2.1. Chemicals and Plant Materials

RPMI-1640 medium, heat inactivated fetal bovine serum

(FBS) and Con A were purchased from Sigma Chemical

Co. (St. Louis, MO, USA). MTT (3-[4,5-dimethylthiazol-

2-yl]-2,5-diphenyl tetrazolium bromide)-based cell pro-

liferation kit was purchased from Boehringer Mann-

heim Gmbh (Mannheim, Germany). All other chemicals

were of analytical grade. Four Dendrolium species,

namely, D. loddigesii Rolfe, D. fimbriatum Hook, D.

nobile Lindl. and D. officinalis Kimura et Migo, were

obtained from an ornamental flora shop in Hong Kong,

and they were authenticated by floral structure with

reference to China Flora. Four samples of Dendrobium

Caulis were randomly bought from local herbal stores.

Voucher specimens of Dendrobium plants and Dendrobii

Caulis have been deposited in the Division of Life

Science, The Hong Kong University o f Science & Tech-

nology (HK UST), Hong Kon g SAR, Chi na.

2.2. Plant/Herbal Extraction

Fresh stems of Dendrobium plants or Dendrobii Caulis

(i.e., dried stems of Dendrobium species) were cut into

small pieces and extracted by 10 volumes (w/v) of dou-

ble distilled water at 60˚C for 2 h. The extraction proce-

dure was repeated once, and the pooled aqueous extracts

were dried by lyophilization. The lyophilized powders

(i.e. Dendrobium extracts) were stored at 20˚C prior to

use for experiment.

2.3. Measurement of in Vitro

Immunopotentiating Activity in

Con A-Stimulated Mouse Splenocytes

Procedures involved in isolation of mouse splenocytes

were conducted under aseptic conditions. Splenic tissue

obtained from adult female ICR mice (25 - 28 g) was

teased with a plastic syringe in a culture dish (60 mm)

containing 10 mL of RPMI-1640 medium, and it was

gently strained through a 200 mesh stainless steel sieve

to remove clumps to produce a cell suspension. The

product was then left to stand on ice for 5 min to remove

tissue fragments. The cell suspension was centrifuged at

600 × g for 10 min an d washed 3 times with RMPI-1 640

medium, and it was finally resuspended in RPMI-1640

medium supplemented with 10% FBS at a concentration

of 1 × 107 viable cells/mL. The viability of isolated

splenocytes, as determined by Trypan blue exclusion test,

was found to be higher than 95%. Mouse splenocytes

were cultured in medium with Con A in the presence or

absence of Dendrobium extract in 96-well microtiter

plates (flat bottom) in a final volume of 100 L. Con A

(prepared in phosphate buffered saline) was added at

final concentrations of 0.5, 1 and 2 g/mL, respectively.

Aliquots of respective Dendrobium extract (10 L in

aqueous solution) were added at increasing final concen-

trations ranging from 1.6 to 100 g/mL. Control wells

were added with 10 L of sterilized double-distilled wa-

ter only. Splenocytes were then cultured for 72 h at 37˚C

in a humidified atmosphere of 5% CO2 in air. At th e end

of the culture period, the extent of splenocyte prolifera-

tion was determined colorimetrically by MTT-based cell

proliferation assay. An aliquot (10 L) of MTT labeling

reagent was added to each well under dark conditions.

After 4 h of incubation, 100 L of solubilization buffer

(10% SDS in 0.01 M HCl) was added, and the mixtures

were incubated in 5% CO2 at 37˚C overnight for dis-

solving the colored crystals. The extent of splenocyte

proliferation was determined by measuring the absorb-

ance at 570 nm using Victor V2 Multi-label Counter

(Perkin Elmer, Turku, Finland). Stimulation index (SI)

was calculated using the equation: SI = mean absorbance

of cells stimulated with Con A/ mean absorbance of cells

not stimulated with Con A. The extent of Con A-stimu-

lated proliferation of isolated splenocytes was estimated

by computing the area under the curve (AUC1) of the

graph plotting stimulatory indices against Con A con-

centrations. Data of AUC1 were expressed as the per-

centage of non-Dendrobium extract-treated control, and

AUC2 was computed from a graph plotting percentages

of control against tested concentrations of Dendrobium

extract. The immunopotentiating activity was expressed

as the difference in AUC2 values between the Dendro-

bium extract-untreated control and Dendrobium-treated

group.

2.4. Measurements of Interleukin-2 (IL-2), IL-6

and Interferon-



(INF-



) Levels

Isolated mouse splenocytes were cultured with Den-

drobium extract (25, 50, 100 g/mL, final concentration)

in the presence or absence of Con A (2 g/mL) for 24 h.

IL-2, IL-6 and INF-



levels in the culture medium was

then measured using ELISA kits (InvitrogenTM Mouse

IL-2 ELISA kit, Invitrogen, Frederick, MD, USA; Invi-

trogenTM Mouse IL-6 ELISA kit, Invitrogen, Camarillo,

CA, USA; ELISAPRO kit for mouse IFN-



, MABTECH,

D. T. W. LAU ET AL.105

Inc., Cincinnati, OH, USA).

3. Results and Discussion

As shown in Figure 1, Con A-stimulated the prolifera-

tion of mouse splenocytes, as indicated by a dose-de-

pendent increase in stimulatory index. While the aqueous

extract of D. officinalis (DO) alone did not produce any

detectable effect on mouse splenocyte proliferation (data

not shown), it caused a dose-dependent enhancement in

Con A-stimulated proliferation of mouse splenocytes in

vitro. When the extent of Con A-stimulated proliferation

was estimated by computing the AUC of the Con A

dose-response curve (AUC1) and then by the AUC of the

DO dose-response curve (AUC2), the extent of Con

A-stimulated proliferation of splenocytes induced by DO

was found to be increased by 23%, when compared with

the DO-untreated and Con A-stimulated control. Among

the tested aqueous extracts of Dendrobium species, D.

nobile (DN) and D. loddigesii (DL) produced a slight

enhancing effect on Con A-stimulated proliferation (1.2

and 3.6%, respectively), whereas D. fimbriatum (DF)

caused a mild suppressive effect (6.4%) (Figure 2).

Among the four tested Dendrobii Caulis samples (DC1-4),

D3 and D4 showed a slight enhancing effect on Con A-

Figure 1. Effect of D. officinalis extract on Con A-stimu-

lated proliferation of mouse splenocytes. Isolated mouse

splenocytes were incubated with increasing concentrations

of Con A (0 - 2 µg/mL) in the absence or presence of an

aqueous extract of D. officinalis (DO) (1.6 - 100 µg/mL), as

described in Materials and methods. Data of two represen-

tative concentrations (6.25 and 100 µg/mL) of DO are

shown. Values given are mean ± S.D., with data obtained

from triplicate assay samples. *Significantly different from

Dendrobium extract-untreated control.

Figure 2. Immunopotentiating activity of Dendrobium ex-

tracts in Con A-stimulated mouse splenocytes. Isolated

mouse splenocytes were incubated with Con A and Den-

drobium extracts (D. fimbriatum, DF; D. loddigesii, DL; D.

nobile, DN; DO; Dendrobium Caulis, DC1-4), as described in

Figure 1. The extent of immunopotentiation was estimated

and data were expressed as Δ AUC2, according to the com-

putation procedure described in Materials and methods.

The value of AUC2 for Dendrobium extract-untreated and

Con A-stimulated control was 10. Values given are mean ±

S.D., with data obtained from triplicate assay samples.

stimulated proliferation of mouse splenocytes (5.4% and

10%), but D1showed undetectable and D2 produced sup-

pressive effect (5.4%).

Splenocyte proliferation is a complex event that in-

volves interaction of cytokines such as IL-1 and IL-2 and

expression of their receptors [29]. The ability of Den-

drobium extract to enhan ce Con A-stimulated splenocyte

proliferation may therefore be mediated by the modula-

tion of cytokine expression and/or function. As shown in

Figure 3(a), Con A caused an increase in IL-2 produc-

tion in cultured mouse splenocytes (Figure 3(b)). DO

enhanced the Con A-stimulated IL-2 production in mouse

splenocytes, with the extent of stimulation being 18% at

100 µg/mL. In addition, Con A also stimulated IL-6 and

INF-



production in mouse splenocytes (Figure 3(b),

(c)), and the Con A-stimulated IL-6 and INF-



produc-

tion were enhanced by DO, with the degree of stimula-

tion being 55% and 24%, respectively, at a concentration

of 100 µg/mL. IL-6 and INF-



, which are cytokines se-

creted by T helper cells, can activate neutrophils [30-32]

and macrophages [33-35], respectively. The enhance-

ment of Con A-stimulated splenocyte proliferation by

DO was associated with the stimulation of cytokine se-

cretion, thereby producing a generalized immunopoten-

D. T. W. LAU ET AL.

106

(a)

(b)

(c)

Figure 3. Effects of D. officincalis extract on cytokine levels

in Con A-stimulated mouse splenocytes. Mouse splenocytes

were treated with Con A (2 µg/mL) and DO (25 - 100 µg/mL).

(a) IL-2, (b) IL-6 and (c) INF-γ evels were measured in the

cultured medium. Values given are mean ± S.D., with data

obtained from triplicate assay samples. *Significantly diffe-

rent from the DO-untreated and Con A-stimulated control.

tiating effect.

An earlier study in our laboratory has shown that

“yin-nourishing” Chinese tonic herbs can enhance the

Con A-stimulated mitogenic response of mouse spleno-

cytes both in vitro and ex vivo [28]. In the present study,

the finding that D. officinalis produced a marked im-

munopotentiating action further supports the use of sple-

nocyte mitogenic assay for the assessment of “yin-nour-

ishing” action. Interestingly, among the tested Den-

drobium species and Dendrobii Caulis samples, D. offi-

cinalis is the most potent in immunopotentiation. The

finding also provides a pharmacological basis for the

adoption of D. officinalis as a premium Dendrobii Caulis

for “yin-nourishing” in the latest edition of Chinese

Pharmacopoeia [12]. The observation that DO obtained

from water extraction at room temperature rather than

60˚C did not produce stimulatory effect on mouse sple-

nocyte proliferation suggests the involvement of poly-

saccharides in immunopotentiating action (unpublished

data).

4. Conclusions

The results indicated that the aqueous extract of D. offi-

cinalis produced immunopotentiating action in mouse

splenocytes, which was more prominent than those of

other tested Dendrobium species and Dendrobii Caulis

samples. The in vitro immunopotentiation assay may be

used for assessing the pharmacological activity of Den-

drobium species. The finding that D. officinalis produ ced

a more potent immunopotentiating action is consistent

with its “yin-nourishing” action in Chinese medicine,

which is more effective than other Dendrobium species

in clinical use.

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