FEBS Letters 581 (2007) 50875093

A Chinese herbal decoction, Danggui Buxue Tang, activates extracellular signal-regulated kinase in cultured T-lymphocytes
Qiu T. Gaoa, Jerry K.H. Cheunga, Jun Lia, Zhi Y. Jianga, Glanice K.Y. Chua, Ran Duana, Anna W.H. Cheunga, Kui J. Zhaoa,b, Roy C.Y. Choia, Tina T.X. Donga, Karl W.K. Tsima,*
a

Department of Biology and Center for Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong b Beijing Friendship Hospital, Aliate of Capital University of Medical Sciences, 95 Yong An Road, Beijing 100050, China Received 31 July 2007; revised 14 September 2007; accepted 24 September 2007 Available online 2 October 2007 Edited by Lukas Huber

Abstract Danggui Buxue Tang (DBT) is prepared from Radix Astragali and Radix Angelicae Sinensis. This Chinese herbal decoction has been shown to stimulate the proliferation of T-lymphocytes; however, the action mechanism of this stimulation has not been revealed. In cultured T-lymphocytes, application of DBT markedly induced the cell proliferation, the release of interleukin-2, -6 and -10, as well as the phosphorylation of extracellular signal-regulated kinases (ERK). The pre-treatment of ERK inhibitor blocked the DBT-induced immune responses. In addition, the polysaccharide-enriched fraction of DBT showed marked responses on the cultured T-lymphocytes suggesting the important role of DBT polysaccharide in triggering such immune responses. 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Danggui Buxue Tang; Radix Astragali; Radix Angelicae Sinensis; T-lymphocyte; Cytokine; ERK

1. Introduction Among thousands of herbal formulae from traditional Chinese medicine (TCM), Danggui Buxue Tang (DBT; a herbal decoction) is a simple combination of only two herbs. DBT was rst described in Neiwaishang Bianhuo Lun by Li Dongyuan in China in AD 1247. Li described the DBT formula should include: 10 qian of Radix Astragali (RA), roots of Astragalus membranaceus (Fisch.) Bunge or Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) P.K. Hsiao, and two qian of Radix Angelicae Sinensis (RAS), roots of Angelica sinensis (Oliv.) Diels. One qian equals about 3 g. The mixed herbs were boiled in two bowls ($500 ml) of water over moderate heat until the nal volume was reduced by half. Traditionally, DBT has been prescribed to women in China as a remedy for menopausal symptoms. These women are directed to drink DBT daily to raise their Qi (vital energy) and nourish their Blood (body circulation). RA has been proven to be an immunostimulant, hepatoprotective, anti-diabetic, analgesic,
* Corresponding author. Fax: +852 2358 1559. E-mail address: botsim@ust.hk (K.W.K. Tsim).

expectorant and sedative drug [1]. On the other hand, RAS is used to invigorate the blood circulation in treating menstrual disorders and to modulate the immune system [2,3]. Although chemical and biological analyses have been performed on the individual herbs [4,5], the rationale for having two herbs in DBT has never been explained; this consequently hinders the development of multi-herb decoctions as disease and disorder remedies. By determining the chemical and biological properties of DBT, the optimized conditions for extraction have been established [4,6], which, interestingly, are in accord with the weight ratio of 5:1 for RA to RAS in the ancient preparation. In addition, a standardized DBT was established by revealing the amounts of RA-derived calycosin and formononetin, and RAS-derived ferulic acid and ligustilide in the decoction [5,7]. The immuno-regulatory property (one of the Qi functions) of DBT was determined here. Previous studies indicated that the proliferation of T-lymphocytes could be stimulated by the treatment of DBT; however, the molecular event responsible for this stimulation has not been determined. To reveal the action mechanism of this ancient herbal decoction, we treated cultured T-lymphocytes with DBT and, subsequently, analyzed the releases of ten cytokines including interleukin (IL)-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, granulocytemacrophage colony stimulating factor (GM-CSF), interferon-c (IFN-c), tumor necrotic factor-a (TNF-a) and the phosphorylation of extracellular signal-regulated kinases (ERK)1/2. In addition, the role of DBT polysaccharide and its signaling pathway for the DBT-induced cytokine release was elucidated.

2. Materials and methods

2.1. Preparation and standardization of DBT Three-year-old A. membranaceus var. mongholicus roots from Shanxi Province [8] and two-year-old A. sinensis roots from Minxian in Gansu Province [3] were collected in 2002. In preparing DBT, exact amounts of RAS and RA were weighed according to a ratio of 5:1 and then mixed well in a vortex. The mixture was boiled in 8 vol of water (v/w) for 2 h and extracted twice; this extraction followed the ancient recipe that had been shown to have the best extracting condition [4,6]. Separate samples of RAS and RA were extracted by the same method. DBT polysaccharides were separated into polysaccharide-enriched fraction and polysaccharide-depleted fraction by precipitating the DBT extract with 70% ethanol. The two fractions were separated after centrifugation at 4000 g for 20 min. The polysaccharide-enriched fraction accounts for $18% of the total dried weight. All the fractions were dried by lyophilization and stored at 80 C.

Abbreviations: TCM, Traditional Chinese medicine; DBT, Danggui Buxue Tang; RA, Radix Astragali; RAS, Radix Angelicae Sinensis; ERK, extracellular signal-regulated kinase

0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.09.053

5088 Standardization of DBT was done by HPLC proling and the contents of ferulic acid, calycosin, formononetin and ligustilide (z-isoform). AR- and HPLC-grade reagents were from Merck (Darmstadt, Germany). A Waters (Milford, MA) HPLC system consisting of a 600 pump, a 717 auto-sampler and a UV/VIS Photodiode Array 2996 Detector was used for all analyses. Chromatographic separations were carried out on a DELTA-PAK C18 column (particle size 4.6 lm, 3.9 mm 150 mm) with acetonitrile (as Solvent A) and: 0.01% phosphoric acid (as Solvent B) in the mobile phase at a ow rate of 1.0 ml/min at room temperature, as described previously [57]. 2.2. T-lymphocyte culture and proliferation assay Human T-lymphocytes were isolated from freshly collected blood of healthy donors by Ficoll-Hypaque [9]. The puried T-lymphocytes were washed twice with RPMI-1640 medium (Invitrogen, Carlsbad, CA) and seeded at a density of 1 105 cells/well in a 96-well culture plate. Six densities of T-lymphocytes (1 1041 106 cells/well) were seeded for making the standard curve of XTT assay. Cultured T-lymphocytes were treated with DBT, RA, RAS, the mixture of RA and RAS, or phytohaemagglutinin (PHA) for 3 days. Then, 1 ml of XTT solution (1 mg/ml; Sigma, St. Louis, MO) was mixed with 5 ll of phenazine methosulfate at 5 mM. Twenty-ve microliters of mixed XTT solution was added to each well, and the cultures were incubated for 4 h in a water-saturated atmosphere of 95% air and 5% CO2. Absorbance at 450 nm was measured. The cell number was determined from the standard curve. In the blocking analyses, MEK inhibitor U0126 (20 lM; Sigma), dissolved in dimethyl sulfoxide (DMSO), was applied 3 h before the application of other drugs. 12O-Tetradecanoylphorbol 13-acetate (TPA; Sigma) at 0.1 lM was used as an ERK activator. 2.3. Cytokine antibody array and ELISA The secretion of dierent cytokines from cultured T-lymphocytes was measured by using Quantibody Human Th1/Th2 Array from Ray Biotech (Norcross, GA). In brief, antibodies against dierent cytokine (IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, GM-CSF, IFNc and TNF-a) were spotted onto the cytokine array by manufacturer. T-lymphocytes were treated with DBT for 3 days. The amount of secreted cytokines was measured by adding 100 ll of conditioned medium onto the array for 2 h. Then, biotin-conjugated primary antibodies were added for 1 h. After washing, Alexa 555-conjugated streptavidin was added for 1 h. The chip was scanned by GenePix Professional 4200A (Synnyvale, CA) at excitation 555 nm and emission 565 nm. The image was analyzed by GenePix Pro 5.0 software program, and the amount of cytokine was quantied according to the standard curve calibrated from the same array. The secreted IL2, IL-6 and IL-10 from T-lymphocytes were further measured by using eBioscience ELISA kit (San Diego, CA). T-lymphocytes were treated with DBT for 3 days. One hundred microliters of conditioned medium in each well was collected, and the amount of IL-2, IL-6 and IL-10 were measured by following the protocol provided from the ELISA kits manufacturer. Finally, the plates were measured at absorbance 450 nm and calibrated. 2.4. Determination of ERK1/2 phosphorylation Cultured T-lymphocytes were treated with DBT for 0, 10, 20, 30, 45, 60 min, respectively. To investigate the eect of inhibitor, the cultures were serum starved with or without the inhibitor for 3 h before the drug applications. After the drug treatments, the cultures were collected immediately in lysis buer (125 mM TrisHCl, 2% SDS, 10% glycerol, 200 mM 2-mercaptoethanol, pH 6.8), and the proteins were subjected to SDSPAGE analysis. Phosphorylated ERK1/2 were recognized by anti-phospho-ERK1/2 and anti-total ERK1/2 antibodies (1:5000; Cell Signaling, Danvers, MA) at 4 C for 12 h, and horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (1:5000; Invitrogen) for 1 h at room temperature. The immuno-complexes were visualized by the enhanced chemiluminescence (ECL) method (GE Healthcare; Piscataway, NJ). The band intensities, recognized by the antibodies in the ECL lm, in control and agonist-stimulated samples were run on the same gel and under strictly standardized ECL conditions. The bands were compared on an image analyzer, using in each case a calibration plot constructed from a parallel gel with serial dilution of one of those samples: this was to ensure the sub-saturation of the gel exposure.

Q.T. Gao et al. / FEBS Letters 581 (2007) 50875093 2.5. Other assays The protein concentrations were measured routinely by Bradfords method with a kit from Bio-Rad Laboratories (Hercules, CA). Statistical tests were made by the PRIMER program, version 1 (Primer of Biostatistics): dierences from basal or control values (as shown in the plots) were classed as signicant (*) where P < 0.05, (**) where P < 0.01 and highly signicant (***) where P < 0.001 by Students t-test.

3. Results 3.1. Standardization of DBT By discovering the amounts of two chemical markers in RA (calycosin and formononetin) and two others in RAS (ferulic acid and ligustilide), we were able to standardize the optimal DBT. The standardized DBT should contain 0.186 mg calycosin, 0.155 mg formononetin, 0.351 mg ferulic acid and 0.204 mg ligustilide per 1 g dried weight of DBT, which was in line with our previous calibration [5,7]. From the extraction eciency calculations, we found that the yield of DBT in this weight ratio was 32 3% (n = 5). These parameters established the chemical standards and the optimal DBT mixture for the remaining biochemical experiments. 3.2. DBT induces T-lymphocyte proliferation In order to reveal the possible immune-regulatory functions of DBT, water extracts of DBT, RA, RAS, and RA + RAS (boiled separately and then mixed together in 5:1 ratio) were applied onto the cultured human T-lymphocytes, and the cell proliferation was determined. All groups showed signicant stimulatory eect on T-lymphocyte proliferation (Fig. 1). The eects of dierent herbal extracts showed a good dosedependent response. At the dosage of 0.1 mg/ml, DBT showed the sub-maximal eect, which was signicantly higher than the other groups. Higher amount of the extracts did not show signicant increase in the cell proliferation, suggesting a saturation eect. The addition of RA + RAS (boiled separately) showed a much lower activation eect on the T-lymphocyte proliferation.

DBT
Proliferation (% of increase)
100 80 60 40 20 0 0 50

RAS RA + RAS

RA

100

150

200

400

PHA

Extract (g/ml)
Fig. 1. Eects of extracts from RA, RAS and DBT in cultured T-lymphocytes. DBT, or RA, or RAS, or RA + RAS at dierent amounts were applied onto cultured T-lymphocytes for 3 days. PHA (10 lg/ml) was used as a positive control. Values of cell proliferation (by XTT assay) are expressed in percentage of increase as compared to control cultures (without herbal extract). Values are in means S.E.M., where n = 5, each with triplicate samples.

Q.T. Gao et al. / FEBS Letters 581 (2007) 50875093

5089

3.3. DBT induces cytokine production in T-lymphocytes Cultured T-lymphocytes were treated for 3 days with extracts derived from DBT, RA, RAS, or RA + RAS. The release of cytokines (IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, GM-CSF, IFN-c and TNF-a) from the drug-treated T-lymphocytes was determined by cytokine antibody array. By comparing to the control cultures, the releases of IL-2, IL-6, IL-8 and IL-10 were markedly induced by DBT (Fig. 2A). Amongst the four cytokines, IL-2, IL-6 and IL-10 showed the specicity for DBT, i.e. not induced by RA, RAS or RA + RAS, and thus they were selected for further analysis. The treatment of DBT caused an increase in the release of IL-2 to $3-fold, IL-6 to $120-fold and IL-10 to $20-fold. Interestingly, the treatment of RA, RAS or a simple mixture of RA and RAS did not change the release of the cytokines suggested that boiling of two herbs together is essential. The levels of IL-4, IL-5, IL-13, GM-CSF, IFN-c and TNF-a were not aected by the treatment of DBT, RA, RAS, and RA + RAS. The releases of IL-2, IL-6 and IL-10 were quantied by an ELISA assay. Fig. 2B and C shows a doseresponse curve for the releases of cytokines after DBT treatment. The responses of the three cytokines were rather similar, i.e. 0.1 mg/ml of DBT in all cases induced the release to a maxi-

mum; the maximal induction for IL-2 was at $4-fold, IL-6 at $20-fold, and IL-10 at $8-fold. By comparing the results from the cytokine array analysis, the results from ELISA assay revealed a lower value, which could be accounted by the sensitivity of the two dierent assay systems. 3.4. DBT induces ERK phosphorylation Activation of ERK, a member of mitogen-activated protein (MAP) kinases that acts as a key signal for T-lymphocyte proliferation, was determined in DBT-treated cells. By using antibodies specic for phosphorylated ERK and total ERK, the phosphorylations of the ERK1 ($44 kDa) and ERK2 ($42 kDa) in cultured T-lymphocytes were revealed: both of the phosphorylations were increased by application of DBT (Fig. 3A and B). The maximal induction of $10-fold on ERK phosphorylation was revealed after 30 min of DBT application. The ERK activation was transient, which fully returned to a low level after 60 min of the treatment. The application of TPA, served as a control, signicantly induced ERK1/2 phosphorylation at a more sustained manner, which is more robust than that of DBT. The DBT-induced ERK phosphorylation at 30 min was fully blocked by pre-treating the cultures with U0126 (20 lM; a MEK inhibitor) (Fig. 3C and D).
150

A
20

DBT RA

Amount of cytokines

4
(x Basal)

***

RAS RA + RAS

100
(x Basal)

Amount of IL-6

50

IL-2

IL-4

IL-5

IL-8

IL-10

IL-13

GM-CSF

IFN-

TNF-

IL-6

B
8

C
IL-10
6
(x Basal)

Amount of cytokines

Amount of cytokine

IL-2

25

IL-6
20
(x Basal)

15 10 5 0

0 0 25 50 75 100 300

25

50

75

100

300

DBT (g/ml)

DBT (g/ml)

Fig. 2. Screening of cytokine release in DBT-treated T-lymphocytes by cytokine array and ELISA assay. DBT, or RA, or RAS, or RA + RAS (all at 0.1 mg/ml) were applied onto cultured T-lymphocytes for 3 days. (A) The conditioned medium (0.1 ml) was loaded onto cytokine array to detect the amount of dierent cytokines. (B, C) The amounts of cytokines (IL-2, IL-6 and IL-10) in the conditioned medium (0.1 ml) were determined by ELISA. Values are expressed as the ratio to basal or control reading where the untreated culture equals to 1, and in means S.E.M., where n = 4. * P < 0.05 and ***P < 0.001 as compared to other groups.

5090

Q.T. Gao et al. / FEBS Letters 581 (2007) 50875093

A
0 Control 10

Min
20 30 45 60
ERK1/2 P ERK1/2 ERK1/2 P

B
ERK P Protein (x Basal)
10 8 6 4 2 0 ERK1/2 P 0 10 20 30 40 50 60

Control DBT

DBT
ERK1/2 ERK1/2 P

RA
ERK1/2

RAS

ERK1/2 ERK1/2 P

Min

RA+RAS

ERK1/2 ERK1/2 P

D
DMSO

TPA
ERK1/2

ERK P Protein (x Basal)

U0126
10 8 6 4 2 0

C
Buffer pre-treatment

tr ol D B T
ERK1/2 P ERK1/2 ERK1/2 P ERK1/2

U0126 pre-treatment

on

Co

nt

DB ro l

Fig. 3. DBT induces ERK phosphorylation in T-lymphocytes. (A) Culture T-lymphocytes were serum starved for 3 h before the addition of DBT (0.1 mg/ml) for dierent time. Total and phosphorylated ERK1/2 were revealed by using specic antibodies. TPA (0.1 lM) was used as a positive control. (B) The quantitation of phosphorylation from the blots in (A) by calibrating the densitometry. (C) The phosphorylation assay was performed on the cultured T-lymphocytes as in (A), except the cultured was pre-treated with U0126 (20 lM) for 3 h before the application of DBT for 30 min. DMSO (a solvent for U0126 at 0.1%) served as a control. (D) The quantitation of phosphorylation from the blots in (C) by calibrating the densitometry. Values are expressed as the ratio to basal reading where time 0 (untreated as basal) equals to 1, and in means S.E.M., where n = 4.

Proliferation / Amount of IL-2 & IL-10 (x Basal)

To further investigate whether the immune-regulatory eects of DBT were related to the ERK1/2 phosphorylation, the release of cytokines in the DBT-treated T-lymphocytes in the present of U0126 was tested. The DBT-induced T-lymphocyte proliferation was fully blocked by U0126 (Fig. 4). In parallel, the releases of IL-2, IL-6 and IL-10, induced by DBT, were blocked by the pre-treatment of U0126 (Fig. 4). These results strongly showed that the pre-treatment of MEK inhibitor could fully abolish the immune-regulatory eects of DBT on T-lymphocytes, and which suggested the crucial role of ERK in mediating the immune responses of DBT. 3.5. The polysaccharide of DBT is an immune activator In order to identify the active component within DBT in triggering the aforementioned immune activations, two fractions: polysaccharide-enriched ($18% of the dried weight) and polysaccharide-depleted ($82% of the dried weight) fractions were obtained. The polysaccharide-enriched fraction, when applied onto the cultured T-lymphocytes, showed a marked increase in its immune activation eect. At 50 lg/ml

Proliferation

IL-2

IL-10

IL-6
25

Amount of IL-6 (x Basal)

8
Buffer
20 15

6 4

U0126

*** **
10 5 0

2 0
D D B T D

Fig. 4. The DBT-induced immuno-modulating eects are mediated by ERK1/2. T-lymphocytes were pre-treated with buer (0.1% DMSO), or 20 lM U0126, for 3 h before the addition of DBT (0.1 mg/ml) for 3 days. The proliferation of T-lymphocyte was assayed by XTT, and the amounts of cytokines were determined by ELISA assay as in Fig. 3. Values are expressed as the ratio to basal reading where the untreated culture equals to 1, and in means S.E.M., where n = 4. **P < 0.01 and ***P < 0.001 as compared to the buer control.

C on tr

B T

on tr

on tr

on

tr

ol

ol

ol

ol

Q.T. Gao et al. / FEBS Letters 581 (2007) 50875093

5091

A
Proliferation (% of increase)
PS-enriched

B
PS-enriched

80 60 40 20 0 0 50 150 200 400 Extract (g/ml) 100

Amount of IL-2 (x Basal)

100

PS-depleted

PS-depleted

6 4 2 0 0 25 50 75 100 300

Extract (g/ml)

C
Amount of IL-6 (x Basal)
30 25 20 15 10 5 0 0 25 50 75 100 300
PS-enriched PS-depleted

D
Amount of IL-10 (x Basal)
10 8 6 4 2 0 0 25 50 75 100 300
PS-enriched PS-depleted

Extract (g/ml)

Extract (g/ml)

Fig. 5. DBT polysaccharide induces the T-lymphocyte proliferation and cytokine release. Cultured T-lymphocytes were treated with polysaccharideenriched (PS-enriched) and polysaccharide-depleted (PS-depleted) fractions for 3 days. (A) Values of cell proliferation (by XTT assay) are expressed in percentage of increase as compared to control cultures (without herbal extract). (BD) The amounts of cytokines (IL-2, IL-6 and IL-10) in the conditioned medium (0.1 ml) were determined by ELISA. Values are expressed as the ratio to basal or control reading where the untreated culture equals to 1. Data were expressed in means S.E.M., where n = 4.

of polysaccharide-enriched fraction, the stimulation of cell proliferation was increased by over 50% that was $5-fold higher than that of polysaccharide-depleted fraction (Fig. 5A). In parallel, the polysaccharides-enriched fraction showed a higher activation eect in stimulating the release of cytokines. At 50 lg/ml of polysaccharide-enriched fraction, the releases of IL-2, IL-6 and IL-10 were increased by $4-fold, $20-fold and $6-fold, respectively (Fig. 5BD). In comparison, the polysaccharides-depleted fraction could only slightly induce the cytokine release. The phosphorylations of the ERK1 ($44 kDa) and ERK2 ($42 kDa) in cultured T-lymphocytes were increased by application of the polysaccharides-enriched fraction (Fig. 6A and B). Similar to the eect of DBT, the maximal induction of $12-fold on ERK phosphorylation was revealed after 30 min of the polysaccharides-enriched fraction of DBT application. The eects of DBT polysaccharides were also fully blocked by pre-treating the cultures with U0126 (Fig. 6C and D). In contrast, the polysaccharides-depleted fraction prepared from DBT did not show activation in the ERK phosphorylation (Fig. 6A and B). The signaling pathway of DBT is similar to that of its polysaccharide, and which indicates the role of polysaccharide in triggering the immune responses. 4. Discussion DBT, an ancient Chinese herbal decoction having a combination of RA and RAS in a weight ratio of 5:1, possesses much

better eect in stimulating cultured T-lymphocytes as compared to that of the extracts derived from RA, or RAS, or RA + RAS (boiled separately and then mixed together in 5:1 ratio). In parallel to this eect, cytokine array analysis revealed a specic set of cytokines including IL-2, IL-6 and IL-10 being stimulated to release from the cultured T-lymphocytes by DBT; the cytokine release was not triggered by RA alone, or RAS alone, or RA + RAS. In addition, the insucient stimulating eect of RA + RAS in cultured T-lymphocytes suggests that boiling of the two herbs together is essential; this method of DBT preparation, indeed, has long been recommended by TCM practitioners. Besides the eect in T-lymphocytes, the role of the correct weight ratio of RA to RAS in making the formulation of DBT has also been demonstrated in cultured breast cancer [7], macrophage [5] and osteoblast [4]. Why does DBT need the boiling of two herbs together and at a ratio of 5:1? The author of DBT, Li Dongyuan, one of the four well-known TCM practitioners in Jin and Yuan Dynasty (the period from 1115 to 1368 A.D.), wrote down the formulation, probably based on accumulated experience in clinical application. The theoretical framework of Chinese medicine is the ve elements: Metal, Wood, Water, Fire and Earth. The ve elements represent dierent organs of our body and work harmoniously to keep the body balance [10]. Li was a great believer of School of Reinforce Earth that is corresponding to the organ of Spleen and the function of Qi; both of them are referring to the immune functions. According to Yijing, a book of ancient Chinese philosophy, Earth represents

5092

Q.T. Gao et al. / FEBS Letters 581 (2007) 50875093

10

20

30

45

60

ERK1/2 ERK1/2 P

PS
ERK1/2 P ERK1/2 ERK1/2 P ERK1/2

Control

PS-enriched
ERK1/2 ERK1/2 P

Buffer pre-treatment

PS-depleted
ERK1/2 ERK1/2 P

U0126 pre-treatment

TPA

ERK1/2

D
12

Buffer U0126

B
12

ERK P Protein (x Basal)

Control PS-enriched PS-depleted

ERK P Protein (x Basal)

10 8 6 4 2 0

10 8 6 4 2

on
C on tr ol

tr

ERK1/2 P

0
0 10 20 30 40 50 60

PS

Min

Fig. 6. DBT polysaccharide induces ERK phosphorylation in T-lymphocytes. The T-lymphocyte cultures were treated with polysaccharide-enriched (PS-enriched at 50 lg/ml) and polysaccharide-depleted (PS-depleted at 50 lg/ml) fractions for 3 days as in Fig. 4. (A) Total and phosphorylated ERK1/2 were revealed by using specic antibodies. TPA (0.1 lM) was used as a positive control. (B) The quantitation of phosphorylation from the blots in (A) by calibrating the densitometry. (C) The phosphorylation assay was performed on the cultured T-lymphocytes as in (A), except the cultured was pre-treated with U0126 (20 lM) for 3 h before the application of the fraction for 30 min. DMSO (a solvent for U0126 at 0.1%) served as a control. (D) The quantitation of phosphorylation from the blots in (C) by calibrating the densitometry. Values are expressed as the ratio to basal reading where time 0 (untreated as basal) equals to 1, and in means S.E.M., where n = 4.

the number of ve. Thus, Li believed that when RA and RAS were combined at a ratio of 5:1, DBT should have the best effect in reinforcing Qi and nourishing Blood for the menopausal symptoms. Two possible hypotheses could explain the unique biological functions of DBT. First, DBT might contain new chemicals or larger amount of active constituents than those in the extracts of RA or RAS alone. The boiling of RA and RAS together might enhance the solubility of new chemicals, and the new chemicals are only soluble in DBT. These additional chemicals could be responsible for the DBT-specic eects. On the other hand, the optimized ratio of the two herbs in yielding more active ingredients can be another good explanation for the DBTspecic eects. The present results suggested that the polysaccharide fraction should contain one of the active ingredients of DBT. In line with that, chemical analyses showed that the content of polysaccharides in DBT was much higher than just adding RA and RAS together [4]. In addition, higher amounts of RA-derived calycosin, formononetin and RAS-derived ferulic acid were also found in DBT decoction [4]. Second, there could be a synergistic eect of dierent components in DBT; this synergistic eect is not present in the extracts of the single herbs. This hypothesis is being supported by the fact that the polysac-

charide-enriched fraction could not fully account the activity of DBT, in particular the enrichment of the activity within the polysaccharide-enriched fraction could not be revealed here. Currently, we still do not have a direct evidence to explain why DBT required the two dierent herbs. DBT possesses signicant eect on T-lymphocyte proliferation, and this eect, possibility, could be a result of the increased releases of IL-2, IL-6 and IL-10. This notion is strongly supported by the eect of MEK inhibitor that blocked the cytokine release as well as the proliferation of T-lymphocyte. Cytokines are a set of soluble proteins that play a key role in immuno-regulation in both innate and adaptive immune responses [11,12]. Among the DBT-specic activated cytokines, IL-2 is an important growth factor for T-lymphocyte, which could amplify lymphocyte responses in vivo [13]. The up regulation of IL-2 by DBT might be one of the important mechanisms for DBTs eect on T-lymphocyte proliferation. IL-10 is a growth factor for B lymphocytes and is responsible for their dierentiation; it can induce immunoglobulin secretion by Blymphocyte [14,15]. Besides, IL-10 is a potent stimulator of NK cells, a function involved in inammation that might contribute to the clearance of the pathogen and facilitate antigen acquisition from dead cells [16]. IL-6 is a multifunctional cyto-

-e nr ic he d
-e nr ic he d

Min

C
ol

Q.T. Gao et al. / FEBS Letters 581 (2007) 50875093

5093 orthogonal array design to optimize the extraction of chemical constituents. Planta Med. 70, 12221227. Gao, Q.T., Choi, R.C., Cheung, A.W., Zhu, J.T., Li, J., Chu, G.K., Duan, R., Cheung, J.K., Jiang, Z.Y., Dong, X.B., Zhao, K.J., Dong, T.T. and Tsim, K.W.K. (2007) Danggui Buxue Tanga Chinese herbal decoction activates the phosphorylations of extracellular signal-regulated kinase and estrogen receptor a in cultured MCF-7 cells. FEBS Lett. 581, 233240. Ma, X.Q., Shi, Q., Duan, J.A., Dong, T.T. and Tsim, K.W.K. (2002) Chemical analysis of Radix Astragali (Huangqi) in China: a comparison with its adulterants and seasonal variations. J. Agric. Food Chem. 50, 48614866. Jain, J., Almquist, S.J., Heiser, A.D., Shlyakhter, D., Leon, E., Memmott, C., Moody, C.S., Nimmesgern, E. and Decker, C. (2002) Characterization of pharmacological ecacy of VX-148, a new, potent Immunosuppressive inosine 5 0 -monophosphate dehydrogenase inhibitor. J. Pharmacol. Exp. Ther. 302, 1272 1277. Ehling, D. (2001) Oriental medicine: an introduction. Altern. Ther. Health Med. 7, 7182. Bellanti, J.A., Kadlec, J.V. and Escobar-Gutierrez, A. (1994) Cytokines and the immune response. Pediatr. Clin. North Am. 41, 597621. Borish, L. and Rosenwasser, L.J. (1996) Update on cytokines. J. Allergy Clin. Immunol. 97, 719734. Nelson, B.H. (2004) IL-2, regulatory T cells, and tolerance. J. Immunol. 172, 39833988. Zuniga-Pfucker, J.C. and Lenardo, M. (1996) Regulation of thymocyte development from immature progenitors. Curr. Opin. Immunol. 8, 215224. Mazur, B., Mertas, A., Sonta-Jakimczyk, D., Szczepanski, T. and Janik-Moszant, A. (2004) Concentration of IL-2, IL-6, IL-8, IL10 and TNF-alpha in children with acute lymphoblastic leukemia after cessation of chemotherapy. Hematol. Oncol. 22, 2734. Mocellin, S., Panelli, M.C., Wang, E., Nagorsen, D. and Marincola, F.M. (2003) The dual role of IL-10. Trends Immunol. 24, 3643. Yamamoto, T., Matsuda, T., Muraguchi, A., Miyazono, K. and Kawabata, M. (2001) Cross-talk between IL-6 and TGF-beta signaling in hepatoma cells. FEBS Lett. 492, 247253. Wu, B.C., Sun, X.F. and Yang, G.Y. (1989) Studies on dierent combination of the Dang Gui Buxue decoction. Chin. Med. Mater. Hei Long Jiang 4, 4145. Li, Y.K., Xu, J. and Zhang, X.C. (1992) Pharmacology study on Dang Gui Buxue decoction. Chin. J. Herb. Pharmacol. Ther. 8, 1620.

kine that plays important roles not only in the immune system, but also in hematopoiesis, and acute phase reactions [17]. In addition to the aforementioned in vitro properties of DBT, the eects of DBT has also been tested and proven in previous animal studies. In DBT-administered mice, the formulation with RA and RAS at 5:1 ratio was the most eective decoction in triggering the immune responses; these responses included the amount of lymphocytes, macrophages and the release of IL-2 [18,19].
Acknowledgement: This research was supported by Grants from the University Grants Committee (AoE/B-10/01) and the Research Grants Council (HKUST6419/06M) of the Hong Kong SAR, and from Jiangsu Key Laboratory for TCM Formulae Research (LTCMF), Nanjing University of Chinese Medicine (FJK 2006011) to K.W.K.T.

[7]

[8]

[9]

[10]

References
[1] Sinclair, S. (1998) Chinese herbs: a clinical review of Astragalus, Ligusticum, and Schizandrae. Altern. Med. Rev. 3, 338344. [2] Wilasrusmee, C., Kittur, S., Siddiqui, J., Bruch, D., Wilasrusmee, S. and Kittur, D.S. (2002) In vitro immunomodulatory eects of ten commonly used herbs on murine lymphocytes. J. Altern. Complement. Med. 8, 467475. [3] Zhao, K.J., Dong, T.T.X., Tu, P.F., Song, Z.H., Lo, C.K. and Tsim, K.W.K. (2003) Molecular genetic and chemical assessment of Radix Angelica (Danggui) in China. J. Agric. Food Chem. 51, 25762583. [4] Dong, T.T.X., Zhao, K.J., Gao, Q.T., Ji, Z.N., Zhu, T.T., Li, J., Duan, R., Cheung, A.W. and Tsim, K.W.K. (2006) Chemical and biological assessment of a Chinese herbal decoction containing Radix Astragali and Radix Angelicae Sinensis: Determination of drug ratio in having optimized properties. J. Agric. Food Chem. 54, 27672774. [5] Gao, Q.T., Cheung, J.K.H., Li, J., Chu, G.K.Y., Duan, R., Cheung, A.W.H., Zhao, K.J., Dong, T.T.X. and Tsim, K.W.K. (2006) A Chinese herbal decoction, Danggui Buxue Tang, prepared from Radix Astragali and Radix Angelicae Sinensis stimulates the immune responses. Planta Med. 73, 12271231. [6] Song, Z.H., Ji, Z.N., Lo, C.K., Dong, T.T., Zhao, K.J., Li, O.T., Haines, C.J., Kung, S.D. and Tsim, K.W.K. (2004) Chemical and biological assessment of a traditional Chinese herbal decoction prepared from Radix Astragali and Radix Angelicae Sinensis:

[11] [12] [13] [14] [15]

[16] [17] [18] [19]