www.elsevier.com/locate/intimp

International Immunopharmacology 4 (2004) 201–211

Immunomodulatory effects of Yun Zhi and Danshen capsules in

health subjects—a randomized, double-blind, placebo-controlled,

crossover study

C.K. Wonga, P.S. Tsea, E.L.Y. Wongb, P.C. Leungb, K.P. Fungb, C.W.K. Lama,*

aDepartment of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kongb Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, NT, Hong Kong

Received 16 September 2003; received in revised form 17 October 2003; accepted 4 December 2003

Abstract

Immunostimulating polysaccharides extracted from the Chinese medicinal plant Yun Zhi (Coriolus versicolor) have been

found to enhance various immunological functions, and Danshen (Salvia miltiorrhiza) to show beneficial effects on the

circulatory system. In the present clinical study, we investigated if regular consumption of Yun Zhi and Danshen capsules could

improve cellular immunity in healthy subjects. One hundred healthy subjects were recruited to take Yun Zhi (50 mg/kg body

weight) plus Danshen (20 mg/kg body weight) or placebo capsules daily for four successive months and, after a 2-month wash-

out period, crossover to take placebo or Yun Zhi plus Danshen capsules for four successive months. Flow cytometry was used to

assess the lymphocyte subtypes and concentration of T helper (Th) cell cytokines in culture supernatant. Gene expression of

cytokines and cytokine receptors of peripheral blood mononuclear cells (PBMC) was analyzed by cDNA expression array.

Results showed that regular oral consumption of Yun Zhi–Danshen capsules could significantly elevate PBMC gene expression

of interleukin (IL)-2 receptor, increase the percentage and absolute counts of T helper cell and ratio of CD4+ (T helper)/CD8+ (T

suppressor and cytotoxic T) cell, and significantly enhance the ex vivo production of typical Th1 cytokine interferon-g from

PBMC activated by phytohemagglutinin and lipopolysaccharide (all p < 0.005). Such consumption had no adverse effects on

liver and renal functions, and the biochemical bone profile. Therefore, regular consumption of Yun Zhi and Danshen could be

beneficial for immunological functions by potential enhancement of cell-mediated immunity in healthy subjects without any

adverse effects.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Yun Zhi; Danshen; T lymphocytes; Interleukin-2 receptor; Interferon-g

1. Introduction

Polysaccharides isolated from different medicinal

plants in traditional Chinese medicine (TCM) have

1567-5769/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.intimp.2003.12.003

* Corresponding author. Tel.: +852-2632-2332; fax: +852-

2636-5090.

E-mail address: waikeilam@cuhk.edu.hk (C.W.K. Lam).

many immunomodulating activities including the en-

hancement of the humoral and cell-mediated immuni-

ty. They have been shown to activate various types of

immune effector cells such as B lymphocytes, T

lymphocytes, cytotoxic T cells (CTL), macrophages,

polymorphonuclear cells, natural killer cells, lympho-

kine activated killer cells and tumor infiltrating lym-

phocytes [1]. Besides the activation of cell-mediated

C.K. Wong et al. / International Immunopharmacology 4 (2004) 201–211202

immunity, many anti-tumor polysaccharides can also

stimulate the in vivo production of cytokines with anti-

tumor activities such as tumor necrosis factor (TNF)-a

[2–4]. Therefore, these polysaccharides may be

immunostimulating and beneficial for enhancing im-

mune functions against neoplasm and infections.

Yun Zhi polysaccharopeptide (PSP) and polysac-

charide Kureha (PSK, Krestin) are a new type of

Biological Response Modifier (BRM) extracted from

the deep-layer cultivated mycelia of the Cov-1 and

CM-101 strains of Yun Zhi (Coriolus versicolor),

respectively. Their active ingredient is a protein-

bound polysaccharide with beta-1,3-glycosidic bond.

They have been used in the BRM therapy of tumors

and enhancement of the immune system. PSP and

PSK have been shown by in vitro studies to possess

anti-tumor activities [5–8], immunorestoration of

CTL and natural killer (NK) cell functions [9], acti-

vation of macrophage [10] and polymorphonuclear

leukocytes [11], enhancement of chemokine and cy-

tokine [e.g. monocyte chemoattractant protein 1 and

interferon (IFN)-g] expression [12,13], and reduction

of oxidative injury [14]. In animal studies, PSP was

found to upregulate the immune response in cancer

radiotherapy [15], restore immunosuppression [16],

and exhibit chemopreventive activity [17,18].

Clinical trials have also shown PSK to be effective

in immunochemotherapy in gastric, esophageal, colo-

rectal, breast and lung cancers [13,19,20]. In phases II

and III double-blind trials in China, the use of PSP

could boost immune cell proliferation, alleviate che-

motherapy symptoms, and enhance tumor infiltration

by dendritic cells and CTL [21]. In a recent clinical

trial, PSP treatment slowed down deterioration of

patients with advanced non-small cell lung cancer

by increasing blood leucocyte and neutrophil counts,

as well as serum IgG and IgM concentrations [22].

Therefore, immunomodulatory activities of Yun Zhi

may have potential benefits on survival and quality of

life, and is compatible with chemotherapy and radia-

tion therapy of cancer.

Danshen (Salvia miltiorrhiza) is a Chinese herbal

medicine that has been widely used for the treatment of

cardiovascular disease [23]. Experimental studies have

shown that Danshen extract can dilate coronary arter-

ies, increase coronary blood flow and scavenge free

radicals in ischemic diseases, thereby reducing the

cellular damage from ischemia and improve heart

function [24,25]. Its active ingredient, sodium tanshi-

none IIA sulfonate, a derivative of tanshinone IIA, can

protect the myocardium from hypertrophy [25]. Apart

from it, Danshen also contains other active ingredients

including protocatechuic aldehyde, tanshinone I, cryp-

totanshinone, hydroxytanshinone, methyltanshinonate,

methylene tanshiquinone, przewatanshinquinone A,

przewatanshinquinone B, miltirone, dihydrotanshi-

none, tanshinol A, tanshinol B etc. A recent study

indicated that Danshen has an anti-hypertensive effect

through the inhibition of angiotensin converting en-

zyme [26]. Therefore, Danshen is beneficial for the

prevention and treatment of cardiovascular disease

[27]. Other studies have shown that Danshen exhibits

chemopreventive effect against alfatoxin B1-induced

hepatocarcinogenesis, inhibit cell growth, and induce

apoptosis in a human hepatoma cell line [28,29].

Moreover, one of the active ingredients of Danshen,

Tanshinone IIA, is a newly confirmed antioxidant that

inhibited the association of lipid peroxidation products

with DNA [30]. Therefore, it is believed that Danshen

can be beneficial for the protection of immune system

and cardiovascular system by scavenging free radicals.

Since antioxidants in diet supplements may have

potential to indirectly improve the immune function in

human beings [31], supplementation of Danshen may

further promote the immunomodulatory activities of

Yun Zhi. We used a combination capsule of Yun Zhi

and Danshen in the present clinical trail to investigate

if regular oral consumption could enhance cellular

immunity in healthy subjects. The induction of T

helper (Th) cytokines and the activation of immune

effector cells were the targeted end points.

2. Materials and methods

2.1. Endotoxin assay

Gram negative bacterial endotoxin level of the Yun

Zhi and Danshen capsule was measured by the QCL-

1000R Chromogenic Limulus Amebocyte Lysate Kit

(Bio-Whittaker, Walkersville, MD, USA).

2.2. Yun Zhi–Danshen capsule

Yun Zhi and Danshen combination capsule and

the matching placebo capsule were supplied by

C.K. Wong et al. / International Immunopharmacology 4 (2004) 201–211 203

Winsor Health Products, Ltd. and were manufactured

conforming to the Good Manufacturing Practice

(GMP) standard. The active drug and placebo capsu-

les were prepared by the GMP qualified Hong Kong

Institute of Biotechnology, Hong Kong using hot

water extraction and lyophilization. The active drug,

Yun Zhi (50 mg/kg body weight, 100% PSP) and

Danshen (20 mg/kg body weight) combination, was

taken orally daily. Placebo capsules were filled with

microcrystalline cellulose and colored to the same

color as the Yun Zhi–Danshen extracts, all identical

in appearance, smell, taste and weight with Yun Zhi–

Danshen capsules.

2.3. Subjects and blood samples

One hundred healthy, mentally alert nonsmoking

subjects (agez 30) not taking any other medication

were recruited. Their medical history was obtained

from a questionnaire survey, and health status ascer-

tained by blood tests (glucose, liver function, renal

function, and complete blood picture) to exclude

common diseases. In this double-blind, placebo-con-

trolled, crossover clinical trial, 100 recruited healthy

subjects were enrolled and randomized into two study

groups (Groups A and B) according to a computer

generated blocked randomization list in a double-

blind fashion. For the subjects of Group A (n = 44),

12 placebo capsules were taken per day for the first

four successive months. The subsequent 2 months was

a wash-out period when neither active capsule nor

placebo was received. Afterwards, 12 active capsules

of Yun Zhi and Danshen mixture were taken per day

for the following 4 months. For the subjects of Group

B (n = 46), 12 active capsules of Yun Zhi and Danshen

mixture were taken daily for the first four successive

months. Neither active capsule nor placebo was re-

ceived in the later 2 months. Afterwards, 12 placebo

capsules were taken per day for the following 4

months. Drug compliance and adverse effects were

recorded. Quality of life was evaluated using a Can-

tonese version of SF-36 Health Survey questionnaire

[32]. Other vitamins, minerals, herbal medicine, alter-

native dietary therapies were excluded of the subjects

during the study period. The protocol was approved

by the Clinical Research Ethics Committee of the

Chinese University of Hong Kong, and written in-

formed consent was obtained from the participants.

2.4. Flow cytometric analysis of CD4+/CD8+/natural

killers (NK)/B cells

The MultiTEST IMK Kit with TruCOUNT Tubes

(Becton Dickinson, California, USA) by the lyse/no-

wash method was used for the assessment of the ratio

and absolute counts of CD4+/CD8+/NK/B cells in

whole blood samples using a four-color FASCalibur

flow cytometer (Becton Dickinson) [33].

2.5. Whole blood assay (WBA)

The method of Viallard et al. [34] was adopted.

After a maximum storage period of 1 h of collected

EDTA blood at room temperature, blood was diluted

1:1 with RPMI 1640 (Gibco Laboratories, New York,

USA), and 0.5 ml aliquots were deposited in each well

of a 24-well plate (Nalge Nunc International, Illinois,

USA). The blood culture was then incubated with or

without phytohaemagglutinin (PHA) (Sigma, Mis-

souri, USA) at 5 Ag/ml and lipopolysaccharide

(LPS) at 25 Ag/ml (Sigma) for 24 h at 37 jC in a

5% CO2 atmosphere. After incubation, the cell free

supernatant was harvested and stored at � 70 jC until

CBA for cytokines. The percentage increase of cyto-

kines released from PBMC of each group after the

incubation with PHA and LPS was calculated as

[(treatment� control)/control]� 100%.

2.6. Cytokine assays using cytometric bead array

(CBA)

Interleukin (IL)-2, IL-4, IL-6, IL-10, TNF-a and

IFN-g in culture supernatant were measured by human

Th1/Th2 cytokine cytometric bead array (CBA) (BD

Pharmingen, California, USA) using flow cytometry

(FACSCalibur, Becton Dickinson) [35]. In CBA, six

bead populations with distinct fluorescence intensities

had been coated with capture antibodies specific for

six different cytokines. These bead populations could

be resolved in the fluorescence channels of the flow

cytometer. After the beads had been incubated with 50

Al of supernatant, different cytokines or chemokines in

the sample were captured by their corresponding

beads. The cytokine/chemokine captured beads were

then mixed with phycoerythrin-conjugated detection

antibodies to form sandwich complexes. Following

incubation, washing and acquisition of fluorescence

C.K. Wong et al. / International Immunopharmacology 4 (2004) 201–211204

data, the results were generated in graphical format

using the BD CBA software. The percentage (%)

change of different cytokines of each subject group

before and after taking capsules was calculated as

[(Month 4�Month 0)/Month 0]� 100% or [(Month

10�Month 6)/Month 6]� 100%.

2.7. cDNA expression array

Peripheral blood mononuclear cells (PBMC) were

isolated from heparinized venous blood by Ficoll-

paque density gradient centrifugation (Amersham

Fig. 1. The (a) percentage of Th lymphocytes (CD3+CD4+) per lymphoc

(CD3+CD4+) per microliter of peripheral whole blood of healthy subjects b

(M2 or M8) or 4 (M4 or M10) months. Results are presented as meanF 2

absolute counts of T helper lymphocytes at different time points. Intraind

assessed by a two-way ANOVA (Multivariate) for repeated measures. Sig

Zhi–Danshen for 4 months ( p= 0.032). M=Month, TCM=Yun Zhi–Da

Pharmacia Biotech, Uppsala, Sweden). Total RNA

was extracted from PBMC using RNeasy Mini Kit

(Qiagen, Hilden, Germany). cDNA expression array

analysis was performed using nonradioactive Human

Inflammatory Cytokine/Receptor GEArrayk Q se-

ries Kit (SuperArray, Bethesda, MD, USA) [36].

Briefly, total RNA was reverse-transcribed with re-

verse transcriptase and GEAprimer mix in the pres-

ence of biotin-16-dUTP. The biotinylated cDNA

probes were denatured and added to the hybridiza-

tion solution. GEArrayk Q-series membranes dotted

with tetra-spot cDNA fragments of 96 human in-

yte population (CD45+) and (b) absolute count of Th lymphocytes

efore (M0 or M6) or after taking Yun Zhi–Danshen or placebo for 2

S.E.M. showing the 95% confidence interval of the percentage and

ividual and interindividual differences at different time points were

nificant differences were found in Groups A and B after taking Yun

nshen.

C.K. Wong et al. / International Immunopharmacology 4 (2004) 201–211 205

flammatory cytokine and receptor genes were pre-

hybridized at 60 jC for 2 h and then hybridized

overnight with the cDNA probes. Membranes were

then washed, blocked and incubated with alkaline

phosphatase-conjugated streptavidin. The labeled bi-

otin on the membrane was detected by chemilumi-

nescent method and the luminescence intensities of

hybridized cDNA probes were analyzed using Bio-

Rad Quantity Onek software (Bio-Rad Laboratories,

California, USA). The percentage change of gene

expression was calculated as [(intensity after treat-

ment� intensity before treatment)/intensity before

treatment� 100%] using glyceraldehyde-3-phosphate

dehydrogenase (GAPDH) as internal control.

2.8. Statistical analysis

Two-way ANOVA (Multivariate) for repeated

measurements was used for the evaluation of the

changes in the clinical laboratory parameters, cytokine

levels, and the percentages and absolute counts of T/

B/NK cells. All analyses were performed using the

Statistical Package for Social Sciences (SPSS) for

Windows, Version 10.0 (SPSS, Illinois, USA). A

probability ( p) < 0.05 was considered as significantly

different.

Fig. 2. The ratio of T helper (CD3+CD4+) and T suppressor plus

CTL (CD3+CD8+) of healthy subjects before (M0 or M6) or after

taking Yun Zhi–Danshen or placebo for 2 (M2 or M8) or 4 (M4 or

M10) months. Results are presented as meanF 2 S.E.M. showing

the 95 % confidence interval of the ratio. Intraindividual and

interindividual differences at different time points were assessed by

a two-way ANOVA (Multivariate) for repeated measures. Signif-

icant differences were found of Groups A and B after taking Yun

Zhi–Danshen for 4 months ( p< 0.001). M=Month, TCM=Yun

Zhi–Danshen.

3. Results

3.1. Dropout of subjects during study

Within the 10 months study period, there were a

total of 18 dropout cases, 3 of them withdrew from

the trial during the first treatment period (before

M2), 5 withdrew during the second treatment period

(before M4), 1 withdrew before the wash-out period

(M6), 2 withdrew during the third treatment period

(before M8) and 6 withdrew during the forth treat-

ment period (before M10). They discontinued study

due to different reasons such as no time for blood

collection, unpleasant feeling in stomach for taking

12 capsules per day or lack of compliance. However,

no serious adverse effect was reported in adverse

events form in the whole study period of all partic-

ipants. The remaining 82 subjects comprised 33 men

and 49 women with a mean (S.D.) age of 42.94

(7.28) years.

3.2. Endotoxin level of the Yun Zhi and Danshen

active capsule

Undetectable endotoxin level was found for the Yun

Zhi–Danshen powder at the concentration of 1 mg/ml.

Since the lowest detection limit from the standard curve

is 0.1 Endotoxin Unit (EU)/ml, the endotoxin concen-

tration of the sample was less than 0.1 EU/mg. This is

Table 1

Differential expression of detectable human inflammatory cytokine or cytokine receptor genes in PBMC of healthy human subjects

Position Gene Density ratio in gene expression level

Group A

(before crossover)

Group B

(before crossover)

Group A

(after crossover)

Group B

(after crossover)

Interleukins and receptors

43 IL2Rb 0.91* (0.70–1.07) 1.28* (0.98–2.28) 1.53** (1.05–2.33) 0.88** (0.74–1.08)

44 IL2Rc 0.95* (0.78–1.09) 1.36* (0.94–1.48) 1.32** (1.00–2.46) 0.91** (0.75–1.01)

49 IL6R 0.91 (0.85–1.23) 1.27 (0.93–2.13) 1.51** (0.98–1.81) 0.72** (0.53–0.98)

50 IL6ST 1.13 (0.69–1.29) 1.07 (0.67–1.59) 1.13 (0.82–2.10) 0.80 (0.70–1.32)

16 IL10Ra 1.00 (0.69–4.35) 1.15 (0.82–1.69) 1.51 (0.98–1.81) 1.01 (0.79–1.54)

17 IL10Rb 0.84 (0.54–2.87) 1.24 (0.63–3.91) 1.67 (1.46–6.23) 0.80 (0.59–7.43)

31 IL16 0.99 (0.82–1.96) 1.44 (0.80–1.67) 1.22 (0.69–2.54) 1.25 (0.77–1.59)

25 IL13Ra1 0.90 (0.47–2.04) 1.11 (0.47–2.87) 1.24 (1.12–2.27) 1.14 (0.63–3.95)

23 IL17R 1.27 (0.57–2.62) 1.58 (0.74–2.04) 1.46 (1.07–4.34) 1.22 (0.73–2.45)

Chemokines

Subfamily A (Cys–Cys)

58 SCYA1 (I-309) 1.03 (0.94–1.15) 1.02 (0.75–1.29) 1.02 (0.90–1.56) 0.99 (0.59–1.91)

75 SCYA4 (MIP-1b) 0.82 (0.48–1.19) 1.03 (0.80–1.97) 1.02 (0.78–1.89) 0.81 (0.53–1.24)

76 SCYA5 (RANTES) 0.65 (0.35–1.59) 1.09 (0.72–1.65) 0.98 (0.79–1.63) 0.88 (0.74–1.34)

64 SCYA17 (TARC) 1.09 (0.56–2.38) 1.14 (0.93–2.06) 0.96 (0.58–1.63) 1.07 (0.53–1.51)

66 SCYA19 (MCP-1) 1.02 (0.40–2.06) 1.21 (0.90–1.76) 0.97 (0.69–1.27) 0.97 (0.69–1.27)

70 SCYA22 (MDC) 1.11 (0.41–1.81) 1.26 (0.88–1.85) 1.15 (0.57–1.57) 0.94 (0.76–1.09)

71 SCYA23 (MPIF-1) 1.24 (0.77–2.09) 1.09 (0.86–1.50) 1.06 (0.92–1.40) 1.18 (0.78–2.46)

73 SCYA25 (TECK) 1.15 (0.64–1.91) 1.34 (1.05–1.93) 1.05 (0.76–1.42) 1.07 (0.92–1.57)

Subfamily B (Cys–X–Cys)

83 SCYB6 (GCP-2) 1.11 (0.48–1.51) 1.44 (1.04–1.77) 0.89 (0.68–1.46) 0.59 (0.12–1.22)

81 SCYB13 (ENA-78/LIX) 1.09 (0.93–1.27) 0.98 (0.76–1.42) 1.19 (1.06–1.73) 1.15 (0.80–2.94)

Subfamily C

85 SCYC2 (SCM-1b) 0.85 (0.44–1.50) 1.43 (0.96–2.09) 0.91 (0.66–1.27) 0.96 (0.78–1.22)

Chemokine receptors

3 CCR2 0.90 (0.72–1.07) 1.21 (0.64–2.51) 1.45 (0.81–2.42) 0.99 (0.74–3.2)

6 CCR5 0.90 (0.61–1.54) 1.10 (0.67–1.64) 1.30 (1.03–5.218) 1.54 (0.52–2.38)

8 CCR7 1.13 (0.79–2.74) 1.34 (0.86–2.27) 1.08 (0.49–2.04) 1.42 (0.65–1.67)

13 CXCR4 0.99 (0.86–1.97) 1.36 (0.84–1.84) 1.55 (0.95–2.74) 0.92 (0.48–1.62)

12 CX3CR1 1.44 (0.80–2.17) 1.28 (0.94–1.66) 0.92 (0.56–1.35) 1.14 (0.82–2.74)

TGF ligands

91 TGFb1 0.94 (0.57–2.04) 1.12 (0.82–2.16) 1.01 (0.36–1.46) 1.01 (0.70–1.45)

93 TGFb3 1.09 (0.63–1.68) 1.22 (0.93–1.98) 1.06 (0.77–1.72) 0.77 (0.49–1.16)

TNF ligand receptors

96 TNFRSF1B (TNFR2) 1.09 (0.85–1.79) 1.02 (0.80–1.40) 1.07 (0.84–1.34) 1.09 (0.93–1.19)

Other related gene

57 MIF 1.06 (0.70–1.66) 1.04 (0.80–1.50) 1.20 (0.98–1.44) 0.82 (0.43–1.19)

Results are shown as median (interquartile range). Intergroup differences at the same time period were assessed by Wilcoxon–Mann–Whitney

U-test.

*p< 0.05.

**p< 0.01.

C.K. Wong et al. / International Immunopharmacology 4 (2004) 201–211206

C.K. Wong et al. / International Immunopharmacology 4 (2004) 201–211 207

much lower than the endotoxin limit for a parenteral

drug (5 EU/kg body weight), i.e. the maximum allow-

able amount of endotoxin that can be administered per

hour without eliciting a pyrogenic response in the body.

For a 70 kg adult, up to 350 EU may be administered

per hour into the body (Associates of Cape Cod,

Massachusetts, USA). Therefore, there was negligible

endotoxin level (i.e. < 500 EU in 5 g Yun Zhi–

Danshen powder for 12 capsules daily) that could

possess any pyrogenic response in human bodies.

Fig. 3. Supernatant concentration and percentage increase of ex vivo IFN

culture supernatant for IFN-g measured by CBA assay was derived from w

and LPS (25 Ag/ml) for 24 h. Results are presented as (A) supernatant co

confidence interval of percentage increase in IFN-g concentrations. Intra

different time points were assessed by a two-way ANOVA (Multivariate) fo

and B after taking Yun Zhi–Danshen for 4 months ( p= 0.006). M=Mon

3.3. Percentage, absolute number and ratio of CD4+/

CD8+/NK/B cells

Fig. 1a shows the percentage of T helper (Th)

lymphocytes (CD3+CD4+) per lymphocyte popula-

tion (CD45+) in peripheral whole blood of healthy

subjects before (M0 or M6) or after taking Yun Zhi–

Danshen or placebo for 2 (M2 or M8) or 4 (M4 or

M10) months. The percentage of T helper lympho-

cytes was significantly higher with the subjects

-g release from mitogen activated-PBMC of healthy subjects. The

hole blood cultured with medium in the presence of PHA (5 Ag/ml)

ncentration (pg/ml) and (B, C) meanF 2 S.E.M. showing the 95%

individual and interindividual differences of percentage increase at

r repeated measures. Significant differences were found of Groups A

th, TCM=Yun Zhi–Danshen.

C.K. Wong et al. / International Immunopharmacology 4 (2004) 201–211208

taking Yun Zhi–Danshen than placebo ( p = 0.027).

Fig. 1b shows the absolute count of T helper

lymphocytes (CD3+CD4+) per lymphocyte microliter

of peripheral whole blood of healthy subjects. The

absolute count of T helper lymphocytes was signif-

icantly elevated ( p = 0.032) when the subjects taking

Yun Zhi–Danshen were compared with those taking

placebo.

Fig. 2 shows the ratio of Th lymphocytes

(CD3+CD4+) and CTL plus suppressor T (Ts) cells

(CD3+CD8+) of healthy subjects before (M0 or M6)

and after taking Yun Zhi–Danshen or placebo for 2

(M2 or M8) or 4 (M4 or M10) months. The ratio was

significantly elevated ( p < 0.001) in subjects after

taking Yun Zhi–Danshen for 4 months. The percent-

age and absolute counts of total lymphocytes

(CD45+), T (CD3+), CTL plus Ts (CD3+CD8+), NK

(CD16+CD56+) and B (CD19+) lymphocytes did not

exhibit any significant change after taking Yun Zhi–

Danshen and placebo.

3.4. cDNA expression array

As shown in Table 1, the density ratio of gene

expression of IL-2 receptor h and g chain was

significantly upregulated in Group B subjects who

had taken Yun Zhi–Danshen capsule for 4 months

when compared with Group Awho had taken placebo

for 4 months before crossover [1.28 (0.98–2.28) vs.

0.91 (0.70–1.07) and 1.36 (0.94–1.48) vs. 0.95

(0.78–1.09, respectively, p < 0.05]. After crossover,

the gene expressions of IL-2 receptor h and g chain

was significantly upregulated in the subjects of Group

A who had taken Yun Zhi–Danshen capsule for 4

months, compared with Group B subjects who had

taken placebo [1.53 (1.05–2.33) vs. 0.88 (0.74–1.08)

and 1.32 (1.00–2.46) vs. 0.91 (0.75–1.01), respec-

tively, p < 0.01].

There was no significant difference in the gene

expression of IL-6 receptor between the subjects of

Group B and that of Group A before crossover [0.91

(0.85–1.23) vs. 1.27 (0.93–2.13), p>0.05]. However,

after crossover, the gene expression of IL-6 receptor

was significantly upregulated in the subjects of Group

A who had taken Yun Zhi–Danshen capsule for 4

months when compared with Group B subjects who

had taken placebo [1.51 (0.98–1.81) vs. 0.72 (0.53–

0.98), p < 0.01].

All other expressed genes listed in Table 1 showed

no significant difference between Group A and Group

B before and after crossover ( p>0.05) (Table 1). For

the remaining genes of other cytokines, chemokines

and receptors that have not been listed in Table 1,

undetectable signal was obtained.

3.5. Ex vivo WBA

In Fig. 3, the percentage increase in ex vivo

production of IFN-g after incubation with LPS and

PHA was significantly higher in Group A (Yun Zhi–

Danshen) when compared with Group B (placebo)

after wash-out period ( p = 0.006). However, there was

no significant difference between the percentage

change of IL-2, IL-4, IL-6, IL-10 and TNF-a before

and after taking Yun Zhi–Danshen or placebo capsu-

les for 4 months.

4. Discussion

Several clinical trials on patients receiving che-

motherapy or radiotherapy have found Yun Zhi PSP

to significantly improve appetite, alleviate weak-

ness, anorexia, vomiting, dryness of throat, sponta-

neous or night sweat and pain, increase weight, and

stabilize white blood cell counts, NK cells, IL-2

and CD4/CD8 ratio [37,38]. In the present study,

the increase in percentage and absolute count of

only CD4+ Th cells in healthy subjects taking Yun

Zhi–Danshen indicated that Yun Zhi–Danshen

could elevate the proliferation of Th cells but not

CTL and T Ts cells. Since Th cells are important

participants in both cell-mediated and humoral

immunity, the increase in proliferation of Th cells

could account, in part, for the immunomodulating

activities in normal human bodies. No effects on Ts

also indicated that Yun Zhi–Danshen does not exert

any immunosuppressive effect on normal subjects.

Moreover, the elevation of CD4+/CD8+ ratio after

regular consumption of Yun Zhi–Danshen capsules

confirmed that the percentage and absolute count of

T helper cells were increased, whereas no signifi-

cant changes were found for CTL and Ts. These

results match the findings in other human clinical

trials showing the increase in CD4+ cells and

CD4+/CD8+ ratio of the stomach, oesophagus and

C.K. Wong et al. / International Immunopharmacology 4 (2004) 201–211 209

lung cancer patients after taking PSP [39,40].

Although we could not observe statistically signif-

icant difference of absolute number and percentage

of NK cells after regular consumption of Yun Zhi–

Danshen capsules, recent data have suggested that

mushroom derived h-glucans alter the maturation of

dendritic cells [41] and enhance in vitro IL-12

production by dendritic cells [42]. However, tanshi-

nones isolated from Danshen inhibit in vitro IFN-g

and IL-12 production [43]. IL-12 and IL-18 have

been demonstrated to potentiate NK and/or natural

killer T cell (NKT)-mediated innate immunity [44];

therefore, whether the Danshen inhibit the activa-

tion of NK cells by Yun Zhi is not certain.

Other study previously demonstrated the induction

of gene expression of IL-8, TNF-a in PBMC after oral

administration of PSK in some of the healthy and

cancer subjects [45]. Our present clinical trial has

demonstrated the elevation of PBMC gene expression

of IL-2 receptor h and g chains in Yun Zhi–Danshen

treated healthy subjects. This is because Danshen has

demonstrated induction effects on sIL-2R; our results

is in concordance with previous report [46]. IL-2, a

Th1 cytokine, is centrally important for the initiation

of adaptive immune response and T cell proliferation

via its autocrine and paracrine functions. Since h and

g chains cooperate to play a role in the IL-2 receptor-

mediated intracellular signals for T cell functions,

such increase of expression may enhance the T cell

response upon activated by future foreign pathogens,

thereby contributing the immunoactivating effect of

the drug.

We have further studied the release of other Th

cytokines from PBMC using WBA. WBA can pre-

serve better the natural environment and constitute an

appropriate milieu for studying in vitro production of

cytokines. It also preserves the natural intercellular

interactions, and circulating stimulatory and inhibitory

mediators including soluble receptors that are present

at their physiological concentrations [35]. Our finding

of significant increase in typical Th1 cytokine IFN-g

release from culture supernatant of PBMC of Yun

Zhi–Danshen treated subjects suggested the activa-

tion of cell-mediated immunity for better defense

against pathogens. Yun Zhi–Danshen can therefore

potentially facilitate the upregulation of the innate

immune response to mitogens, and hence other for-

eign pathogens.

However, there was no gene upregulation of IFN-g

while its production was increased from PBMC of the

Yun Zhi–Danshen treated healthy subjects under

mitogen stimulation. Therefore, under normal and

unstimulated state, the PBMC gene expression of

IFN-g is kept at undetectable level even after regular

consumption of Yun Zhi–Danshen capsules for 4

months. Except genes for IL-2 receptor and IL-6

receptor, regular consumption of Yun Zhi–Danshen

did not upregulate or downregulate other inflamma-

tory related cytokine, chemokine and receptor gene

(Table 1) as well as plasma level of Th1/2 cytokines

IL-2, IL-6, IL-4, IL-10, TNF-a and IFN-g (data not

shown), it further illustrated that Yun Zhi and

Danshen could not cause any inflammatory response

in normal human bodies and thus no change in the

gene expression and plasma concentration of these

cytokines, chemokines and receptors under normal

and unstimulated condition. However, it potentially

increases the response of lymphocyte mediated im-

munity upon stimulation against pathogens as illus-

trated from the results of WBA.

We found that the Yun Zhi–Danshen capsule could

improve the quality of life of the subjects by enhanc-

ing physical function and healthy transition (respond-

ents’ amount of change in their health in general over

a 1-year period) without any adverse side effects using

SF-36 health survey questionnaire. The liver function

(serum total protein, albumin, total bilirubin, alkaline

phosphatase and alanine transaminase), renal function

(serum potassium, sodium, urea, creatinine), bone

profile (serum calcium, phosphate), hematological

parameters (complete blood count, differential blood

count) and clotting factor (fibrinogen) were all within

the normal range before and after Yun Zhi–Danshen

or placebo treatment ( p>0.05, data not shown). In

case of concomitant use of botanical candidates alone

or with prescription medication, combined pharmaco-

kinetic profiles must be considered. It is because

recent in vitro study demonstrated Danshen could

increase metabolic activity of cytochrome P450 iso-

forms, thereby implying drug–herb interactions

[47,48]. Single blood sampling to screen for effects

of botanical combination on the activity of the com-

mon drug metabolizing enzyme isoforms require

investigation.

In conjunction with its in vivo and ex vivo immu-

nomodulatory activities, our present clinical trail fur-

C.K. Wong et al. / International Immunopharmacology 4 (2004) 201–211210

ther strengthens that the regular consumption of Yun

Zhi–Danshen is beneficial for potentiating and mod-

ulating the cell-mediated immunity in healthy subjects

through the elevation of number and functions of Th

cells, but it does not elicit any inflammatory response.

As a result, these shed light to the potential clinical

use for immunomodulatory and anti-tumor functions

of these medicinal herbs. In an attempt to further

elucidate the immunopotentiation and anti-cancer ac-

tivity of Yun Zhi–Danshen capsule, we have been

conducting clinical trials on cancer patients in whom

immune system was suppressed by chemotherapy and

radiotherapy.

Acknowledgements

This study was supported by the Innovation and

Technology Fund, Hong Kong and Winsor Health

Products Ltd.

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