The screening and isolation of an effective anti-endotoxin monomer from Radix Paeoniae Rubra using affinity biosensor technology

International Immunopharmacology 5 (2005) 1007 – 1017 www.elsevier.com/locate/intimp

The screening and isolation of an effective anti-endotoxin monomer from Radix Paeoniae Rubra using affinity biosensor technology Lv Genfaa, Zheng Jianga,T, Zhou Hongb,T, Zheng Yiminc, Wang Liangxib, Wei Guoa, Huang Minga, Jiang Donglena, Wei Lizhaoa a

Medical Research Center, Southwestern Hospital, Third Military Medical University, Chongqing 400038, China b Department of Pharmacology, Third Military Medical University, Chongqing 400038, China c Chongqing Academy of Chinese Materia Medica, Chongqing 400038, China Received 29 November 2004; received in revised form 7 January 2005; accepted 19 January 2005

Abstract Lipopolysaccharide (LPS) is a known trigger in the pathogenesis of sepsis, lipid A being the toxic component. One of several adjuvant therapeutic approaches for severe sepsis is currently focusing on the neutralization of LPS. In order to obtain the components from traditional Chinese herbs that can neutralize the endotoxin, aqueous extractions were tested using affinity biosensor technology. From amongst 42 herbs, eight were found to possess lipid A-binding abilities. Radix Paeoniae Rubras had the highest lipid A-binding ability; therefore an aqueous extraction from this plant was investigated further. After preparation using standard methods, including silica gel chromatography and HPLC, we obtained 1, 2, 3, 4, 6-h-dpentagalloylglucose (PGG), with lipid A-binding ability. It was found that in vitro, PGG directly bound to lipid A, with a Kd of 32 AM, and that it neutralized the endotoxin both in the Limulus Amebocyte Lysate (LAL) assay and in a TNF-a release experiment, in a dose-dependent manner. In in vivo experiments, PGG was found to protect mice from a lethal challenge by LPS, and significantly decreased the plasma endotoxin level both in endotoxemic mice and rats, the reduction of the endotoxin level in rats being tightly associated with the TNF-a level. In conclusion, we demonstrate the effectiveness of affinity biosensor technology in discovering useful agents amongst traditional Chinese herbs and using this approach we found a new antiendotoxin agent. D 2005 Elsevier B.V. All rights reserved. Keywords: Traditional Chinese herbs; Affinity biosensor; Lipopolysaccharides; 1, 2, 3, 4, 6-h-d-pentagalloylglucose; Polymyxin B

1. Introduction T Corresponding authors. Zheng Jiang is to be contacted at Tel.: +86 23 68754435; fax: +86 23 65460584. Zhou Hong, Tel.: +86 23 68752266; fax: +86 23 68752266. E-mail addresses: [email protected] (Z. Jiang)8 [email protected] (Z. Hong). 1567-5769/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2005.01.013

Sepsis results in the activation of numerous proinflammatory mediators such as TNF-a, IL-6 and IL12, which may damage cells and lead to organ injury [1]. Severe sepsis and septic shock are life threatening

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complications of infections and a common cause of death in intensive care units. Recent US and European surveys estimate that severe sepsis accounts for 2– 11% of all admissions to hospitals or intensive care units. Despite improved supportive care, the hospital mortality rate from severe sepsis and septic shock (30% and over 60%, respectively) has not changed significantly over the past several decades [2]. Unfortunately, many experimental inflammatory antagonist-based therapies have failed in sepsis trials, and there are currently few effective adjuvant therapies in clinical use except activated protein C focusing on the coagulation system. Thus it is important to investigate additional inflammatory antagonist-based treatments in the hope of identifying a clinically relevant adjuvant. The lipopolysaccharide (LPS) is a principal component of the outer membrane of gram-negative bacteria, and is a common trigger of sepsis. One of several adjuvant therapeutic approaches for severe sepsis is currently focusing on endotoxin [3,4]. Lipid A, an evolutionarily conserved region of LPS, has been identified as the toxic component of LPS and therefore presents an ideal target against which newly developed drugs may be directed [5,6]. Accordingly, agents that bind to LPS and neutralize its activities could have potential clinical application. In traditional Chinese medical theories, sepsis is a disease classified as bseasonal febrile diseaseQ (wenbing in Chinese) or as bpyrexia symptomQ (re-zheng in Chinese). In this type of approach, the treatment principle is to eliminate fever and toxic materials from the body, using traditional Chinese herbs. There is a large range of herbs available, for example Houttaynia Cordata Thanb, Herba Artemisiae Annuae, and Radix Paeoniae Rubra, are three that have been used to treat wen-bing and re-zheng for thousands of years. It has been reported that many herbs indeed possess antiendotoxin properties [7,8]. However, herbs possess a large number of complex constituents and it is extremely difficult to identify which is the antiendotoxin component. In the present study, we first undertook to confirm the presence of anti-endotoxin component(s) in a panel of 42 traditional Chinese herbs, using affinity biosensor technology. The herb, Radix Paeoniae Rubra, was selected from this panel, and components were then isolated that possessed the highest

lipid A-binding ability. Finally, the component isolated from Radix Paeoniae Rubra was examined for its anti-endotoxin activity both in vitro and in vivo.

2. Materials and methods 2.1. Materials 2.1.1. Reagents LPS (from Escherichia coli O111:B4), Lipid A (from Samonella Re595), MTT, and Polymyxin B sulfate salt (PMB) were purchased from Sigma Chemicals (St Louis, MO, USA). Human TNF-a and IL-6 ELISA kits were from Biosource International (Camarillo, CA, USA). The rat TNF-a ELISA kit was from Diaclone Research (Besanc¸on, France). The kinetic turbidimetric Limulus amebocyte lysate (LAL) kit was from Zhanjiang A and C Biological (Zhanjiang, China). Silica gel was from QingDao Marine Chemical Factory (QingDao, China). 2.1.2. Traditional Chinese herbs Forty-two traditional Chinese herbs (see Table 1) were purchased from Sichuan Province, and identified in the Chongqing Academy of the Chinese Materia Medica (Chongqing, China). 2.1.3. Animals One hundred and 40 Balb/c mice (4–6 weeks old) and eight male Wistar rats (3 months old) were obtained from the Experimental Animal Center of the Third Military Medical University (Chongqing, China). Equal numbers of male and female mice were used. The weight of the mice on the day of the experiments was 20.4 F 3.0 g, and that of the rats was 207.2 F 12.3 g. 2.1.4. Isolation of human peripheral blood mononuclear cells (hPBMC) hPBMC were isolated from healthy volunteers using standard methods [9]. Briefly, after obtaining informed consent from the donors, blood was collected by venipuncture, anticoagulated with 5 U pyrogen-free heparin/ml blood and centrifuged at 500
g for 15 min. The leukocyte-rich buffy coat was then subjected to Ficoll-Hypaque density gradient

L. Genfa et al. / International Immunopharmacology 5 (2005) 1007–1017 Table 1 List of the traditional Chinese herbs examined in this study Name Aconitum Carmichaeli Debx Belaminda Chineusis

Folium Isatidis

Fructus Aurantii Immaturus Centiana Scabra Bge Fructus Cnidii Calvatia Lilacina Fructus Gardeniae Calvatia Gigantea Lbyd Fruetus Forsythiae Chinese Lobelia Herb Chinese White Olive Colla Corii Asini Cortex Phellodendri Colla Corii Asini Cortex Eucommiae

Cortex Cinnamomi Densefruit Pittany Root-Bark Flos Lonicerae

Fructus Mume Herba Andrographitis Herba Artemisiae Annuae Herba Hedyotidis Herba Saussureae Houttaynia Cordata Thanb Iphigenia Iindica Kunthet Monimopetalnm Chinese Peony Root

Pria Cocos Wolf Radix et Rhizoma Rhei Radix Isatidis Radix Pulsatillae Radix Salviae Miltiorrhiizae Radix Scutelariae Radix Sophorae Flavescentis Rhizoma Anemarrhenae Rhizoma Coptidis Rhizoma Smilacis Glabrae Spica Prunellae

Spohora Tonkinnesis Gapnep Viola Yedoensis Makino Whiteflower Patrinia Herb

centrifugation followed by collection of PBMC from the light density fraction. Cells were washed three times with ice-cold PBS before being counted using a hemocytometer and resuspended at the desired density (1.5
106 cells/ml) in RPMI 1640. 2.2. Methods 2.2.1. Screening of LPS-binding agents from Chinese herbs using affinity biosensor technology 2.2.1.1. Assays for binding to lipid A. Lipid A was immobilized on the surface of a hydrophobic cuvette according to the manufacturerVs instructions (Thermo Labsystem, USA), and as described previously [10]. Forty-two herbs were each soaked in water for 2 h, after washing thoroughly with distilled water, and then boiled in water at 100 8C for 45 min. After filtration, the material was centrifuged at 4000
g for 30 min and the supernatants collected, and are termed here baqueous extractionsQ. Five microlitres of aqueous extraction from each herb was added into a cuvette containing 60 Al PBS/

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AE. After 5 min, the cuvette was washed seven times with 60 Al PBS/AE and alternately washed with 0.1 M HCl, PBS/AE, and 10 mM NaOH, respectively. Data analyses were performed using the FASTplot software package (Thermo Labsystem, USA). 2.2.1.2. Capacity of aqueous extractions for binding to LPS. To determine the exact capacity of the aqueous extraction for binding to LPS, we designed an experiment using the Affinity Sensors IAsys Plus system. Four aqueous extractions from Radix Paeoniae Rubra, Chinese White Olive, Radix et Rhizoma Rhei, and Fructus Gardeniae, which possessed the highest binding activity to lipid A, were selected for further study. The details of the experiment were as described previously [10]. Briefly, the aqueous extraction was first pre-incubated with LPS. Provided the active molecules from the aqueous extraction did not saturate the LPS in the pre-incubation solution, then free active molecules could bind to the lipid A immobilized on the cuvette, then would then create a signal that indirectly represented the ability of the aqueous extraction to bind to LPS. Based on the above, hydrophobic cuvettes were coated with immobilized with lipid A. Next, 50 Al of the aqueous extraction was pre-incubated with 50 Alof 10 ng/ml of LPS for 30 min, with 50 Al of the aqueous extraction mixed with 50 Al PBS/AE as a control. Five microlitres of the mixture was added into the cuvette, and data were collected and analyzed using FASTplot software. 2.2.2. Isolation LPS-binding components from Radix Paeoniae Rubras An aqueous extraction from Radix Paeoniae Rubras was obtained as described as above. The pH was titrated to pH 8.0 using 1M NaOH and after 30 min, the solution was centrifuged at 4000
g for 30 min, and the precipitate collected. The precipitate was re-suspended in PBS (pH 6.0), and then titrated to pH 6.2 with 1 M HCl. It was then centrifuged at 4000
g for a further 30 min and the supernatant collected and ¨¨CHI Rotavaconcentrated by rotary-evaporation (BU por R205, Switzerland). The powder obtained was subjected to silica gel column chromatography with elution with methanol/ethyl acetate (2:1, by vol.). Each fraction was assayed for its binding activity to lipid A. The fraction with the highest activity was further purified by HPLC (Varian PrepStar USA). The

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purity of the fraction with the highest activity was 99.8%, and its structure was then analyzed by infrared, mass spectrometry and NMR (nuclear magnetic resonance) at the Chinese Academy of Military Medical Sciences (Beijing, China).

2.2.4. In vivo studies

2.2.3.3. Inhibition cytokine release induced by LPS. hPBMC (1.5
106) were grown in a 48-well plate and incubated for 4 h. Next the monomer, ranging from 10 Ag/ml to 80 Ag/ml in PBS, was added along with 100 ng/ml of LPS. After incubation for another 3 h, the levels of TNF-a and IL-6 in the supernatants were analyzed using the appropriate ELISA kits.

2.2.4.1. Experimental protocol and monomer treatment of mice. The protection of the monomer in mice challenged with lethal LPS was first determined. One hundred and 20 mice were randomly divided into 6 groups (20 mice/group) that were intravenously injected as follows: LPS (20 mg/kg) in group 1, the monomer alone (80 mg/kg) in group 2, LPS (20 mg/kg) plus the monomer (20 mg/kg) in group 3, LPS (20 mg/kg) plus the monomer (40 mg/ kg) in group 4, LPS (20 mg/kg) plus the monomer (80 mg/kg) in group 5, and LPS (20 mg/kg) plus PMB (1 mg/kg) in group 6. The total injection volume was 0.2 ml per 20 g bodyweight. The general conditions and mortality of the mice were observed for 7 days, which were fed ad libitum during the experiment. Next the neutralization of the endotoxin property of the monomer in endotoxemic mice was determined. Twenty mice were randomly divided into five groups (4 mice/group) that were intravenously injected as follows: LPS (50 Ag/kg) in group 1, LPS (50 Ag/kg) plus the monomer (1 mg/kg) in group 2, LPS (50 Ag / kg) plus the monomer (2 mg/kg) in group 3, and LPS (50 Ag/kg) plus the monomer (4 mg/kg) in group 4, and only saline in group 5 (control), The total injection volume was 0.2 ml per 20 g bodyweight. Mice were sacrificed 0.5 h after injection of LPS and blood samples were collected from the heart. Five microlitres of the plasma was diluted in 195 Al of saline. The endotoxin level in the diluent was assayed by the LAL test.

2.2.3.4. Cell viability assay. To exclude the possibility of the monomer affecting the cell viability, an MTT assay was performed. hPBMC (1.5
106cells/ ml, 0.2 ml) were grown in 96-well plates and incubated for 4 h, and the monomer (ranging from 10 Ag/ml to 80 Ag/ml in PBS) was added. After incubation for 2 h, the cells were washed with PBS twice, and 100 Al of fresh medium and 10 Al MTT (5 mg/ml) added into each well for a further 3 h incubation. After centrifugation at 1000 rpm for 5 min, the supernatant was removed and 150 Al of DEMSO added into the wells. MTT crystals were completely solubilized after 10 min and the OD value was then determined at 490 nm.

2.2.4.2. Determination of plasma endotoxin and serum cytokine in rats. Rats were surgically implanted with cardiac catheters to facilitate infusion of sublethal doses of LPS [10]. For these experiments, eight rats were randomly divided into two groups (n=4). Group 1 was given 2 mg/kg LPS, group 2 was given 40 mg/kg of the monomer followed by infusion with LPS. Blood samples (0.3 ml) were drawn 0, 0.5, 1, 2, 4, 8, 12 and 24 h after inception of the experiment and the serum was stored at -80 8C for subsequent TNF-a assay. Meanwhile 10 Al of the plasma was diluted with 990 Al of saline twice, and the solution incubated in 90 8C water for 10 min in order to inactivate heparin. The endotoxin

2.2.3. In vitro studies 2.2.3.1. Affinity assessment. The Kd value for the lipid A monomer was measured as described previously [10]. In brief, seven concentrations of the monomer (8, 4, 2, 1, 0.5, 0.25, 0.125 AM) were added to a cuvette immobilized with lipid A, respectively. A binding curve was generated, and Kd values were generated using the FASTplot and FASTfit software packages (Thermo Labsystem, USA). 2.2.3.2. Neutralization of endotoxin. The ability of the monomer to neutralize LPS was assayed using the LAL test. Different concentrations of the monomer (0–8 Ag/ml) were incubated with LPS (2 ng/ml) at 37 8C for 30 min. Subsequently, 100 Al of this mixture was added to an equal volume of the LAL reagent. The kinetic turbidity was measured using a Tube Reader ATi-321 (Lab Kinetics, United Kingdom).

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Water Extracts Binding to Lipid A 1800

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3. Results 3

3.1. Eight herbs were identified as possessing lipid Abinding activity

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Among the 42 Chinese herbs examined, eight herbs were found to possess lipid A-binding activities (RU N 100 arc second) (Fig. 1). They were Belaminda Chineusis, Colla Corii Asini, Radix Paeoniae Rubras, Radix et Rhizoma Rhei, Fructus Aurantii Immaturus, Chinese White Olive, Fructus Gardeniae and Aconitum Carmichaeli Deb. However, there were large differences among the herbs (response units ranging from 125.35 to 1204.1 arc second), which suggested

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Fig. 1. The aqueous extractions from eight traditional Chinese herbs have lipid A-binding ability. Five microlitres of an aqueous extraction from each herb was placed into a cuvette containing 60 Al PBS/AE. After 5 min, the cuvette was washed seven times with 60 Al PBS/AE and alternately washed with 0.1 M HCl, PBS/AE, and 10 mM NaOH, respectively. Data analyses were performed using FASTplot software. Other details are as described under Methods.

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level in the solution was assayed using the LAL test. 2.2.5. Statistics and presentation of data The Chi-squared exact test was used to analyze for the significance of differences in mouse mortality among the groups. Cytokine concentrations are expressed as mean F std.dev. Each experiment was repeated at least twice and each data point represents the mean of at least three parallel samples. A StudentVs t-test was used to examine the differences in cytokine and endotoxin concentrations. A p value of less than 0.05 was considered significant, and a value less than 0.01 was considered highly significant.

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Time (minutes) 1. Radix Paeoniae Rubras 2. Radix Paeoniae Rubras + LPS 3. Radix et Rhizoma Rhei 4. Radix et Rhizoma Rhei+ LPS 5. Chinese White Olive 6. Chinese White Olive + LPS 7. Fructus Gardeniae 8. Fructus Gardeniae + LPS

Fig. 2. LPS binding capacity to aqueous extractions. Fifty microlitres each from the aqueous extraction from four traditional Chinese herbs (Radix Paeoniae Rubra, Chinese White Olive, Radix et Rhizoma Rhei, and Fructus Gardeniae) were pre-incubated with 50 Al of 10 ng/ml of LPS for 30 min, and 50 Al of the aqueous extraction was mixed with 50 Al PBS/AE was as control. Five microlitres of the mixture were placed into a cuvette in order to allow binding to Lipid A. Other details are as described under Methods.

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endotoxin. It was therefore used for extraction and purification of the anti-endotoxin agent. 3.2. Isolation of 1, 2, 3, 4, 6-b-d-pentagalloylglucose (PGG) from Radix Paeoniae Rubras After the structure was analyzed by infrared, mass spectrometry and NMR (data not shown), the monomer with binding activity for lipid A was confirmed as 1, 2, 3, 4, 6-h-d-pentagalloylglucose (PGG) (Fig. 3). Additionally, another two monomers were also found but the structures were not confirmed yet. 3.3. In vitro studies Fig. 3. Structure of 1, 2, 3, 4, 6-h-d-pentagalloylglucose (PGG).

there were significant differences in the anti-LPS activities among these herbs. From a curve of the capacity of aqueous extractions binding to lipid A (Fig. 2), it can be seen that the aqueous extraction from Radix Paeoniae Rubras had the highest response compared with the other three extractions, indicating that it possessed a higher binding capability with lipid A, after incubation with

3.3.2. PGG inhibits the LPS-induced LAL reaction PGG was tested in the LAL assay for its ability to neutralize LPS. PGG neutralized endotoxin in a dosedependent manner (Fig. 5), as follows: PGG in 2, 4 8µM

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3.3.1. PGG possessed a high affinity for lipid A To define the specificity of PGG binding to lipid A, FASTplot was used to generate binding curves and the Kd value was generated by FASTfit. From Fig. 4 it can be seen that PGG bound lipid A with a Kd of 3.2 AM.

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Time (minutes) Intercept = 0.02897852 Gradient = 8.995375e+3 Kd = Intercept/Gradient = 3.2e-6M Fig. 4. Binding curve of PGG to lipid A. Five microlitres of PGG (8, 4, 2, 1, 0.5, 0.25, and 0.125 AM) was placed into a cuvette containing 60 Al PBS/AE. After 5 min, the cuvette was washed seven times with 60 Al PBS/AE and alternately washed with 0.1 M HCl, PBS/AE, and 10 mM NaOH, respectively. Data analyses were performed with the FASTplot software. Other details are as described under Methods.

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PGG (µg/kg) Fig. 5. PGG neutralizes LPS in vitro. Different concentrations of the monomer (0–8 Ag/ml) were incubated with LPS (2 ng/ml) at 37 8C for 30 min. Subsequently, 100 Al of this mixture was added to an equal volume of the LAL reagent. The kinetic turbidity was measured using a ATi-321 tube reader. Data are the means F s.d. of triplicate determinations. The experiment was done in triplicate. *p b 0.05 compared with LPS; **p b 0.01 compared with LPS. Other details are as described under Methods.

Fig. 6. PGG inhibited TNF-a and IL-6 release from LPS-stimulated hPBMC. hPBMC (1.5
106) were grown in 48-well plates and incubated for 4 h. The monomer, ranging from 10 Ag/ml to 80 Ag/ml in PBS was added, then 100 ng/ml of LPS was added. Incubation was carried out for another 3 h, the level of TNF-a and IL-6, in the supernatants, was analyzed using the appropriate ELISA kits. Data are the means F s.d. of triplicate determinations. The experiments were performed in triplicate. *p b 0.05 compared with LPS; **p b 0.01 compared with LPS. Other details are as described under Methods.

and 8 Ag/ml, neutralized 35.8?, 75.4% and 78.5% of LPS in 2 ng/ml, respectively. This indicated its high potency for endotoxin neutralization at the higher concentrations.

PGG pre-treatment), all died within 24 h. By contrast, animals pretreated with PGG were protected from LPS-induced lethality, in a dose-dependent manner; PGG (40 mg/kg) protected mice to a significant

3.4. In vivo studies 3.4.1. Protecting mice from an LPS challenge To test whether PGG could protect mice from a lethal challenge with LPS, test animals were injected with different concentrations of PGG, prior to a lethal challenge with LPS (20 mg/kg). For the endotoxemic mice, among 20 mice challenged by LPS (without

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3.3.3. PGG inhibits LPS-stimulated TNF-a and IL-6 release from hPBMC In order to confirm that the PGG inhibition of the LPS-induced LAL reaction was paralleled with an inhibitory activity on cytokine release, the ability of PGG to inhibit TNF-a and IL-6 secretion from hPBMC was determined. From Fig. 6, it can be seen that PGG inhibited TNF-a and IL-6 in a dosedependent manner: 80 Ag/ml of PGG potently inhibited 61.4% of TNF-a and 42.5% of IL-6 release ( p b 0.01). The inhibition on TNF-a and IL-6 secretion was not related to its influence on the cell viability (Fig. 7) at least for concentration range from 10 to 80 Ag/ml.

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PGG (µg/ml) Fig. 7. Influence of PGG on cell viability using the MTT assay. hPBMC (1.5
10 6 cells/ml, 0.2 ml) were grown in a 96-well plate and incubated for 4 h. PGG, ranging from 10 Ag/ml to 80 Ag/ml in PBS, was then added. After incubation for 2 h, the cells were washed with PBS twice, then 100 Al fresh medium and 10 Al MTT (5 mg/ml) were added into each well and incubated for a further 3 h. After centrifugation at 1000 rpm for 5 min, the supernanant was removed and 150 Al of DEMSO was added into the well. MTT crystals were completely solubilized by vibrating for 10 min. The OD value was determined at 490 nm. Data are the means F s.d. of nine wells. The experiment was performed in triplicate. zp N 0.05 compared with treatment without PGG. Other details are as described under Methods.

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PGG significantly reduced the plasma endotoxin level (significant at p b 0.05, see Fig. 10A), as early as 30 min after the LPS challenge, and the effect lasted for 2 h. Rats treated with LPS showed large increases in TNF-a, which peaked 1 h after the LPS treatment (297.9 F 75.2 pg/ml). However, pretreatment with 40 mg/kg PGG significantly reduced the peak level of TNF-a (101.1 F 40.4 pg/ml) (Fig. 10B). These results suggest that PGG neutralizes endotoxin, not only in vitro but also in vivo, since cytokine release is tightly related to plasma endotoxin. Taken together, our results show that PGG could potently suppress LPS-induced TNF-a release (an early cytokine) in vivo. The levels of endotoxin paralleled those of TNF-a.

degree ( p b 0.05) and PGG (80 mg/kg) had a highly significant protective effect ( p b 0.01) (Fig. 8). 3.4.2. Neutralization of endotoxin in endotoxemic mice When the plasma endotoxin level in endotoxemic mice was examined, for a PGG ranging from 2 to 4 mg/kg, all showed a significant dose-dependent reduction in the plasma endotoxin level. The extent of the reduction was very significant for the group of 4 mg/kg ( p b 0.01) (Fig. 9). 3.4.3. Neutralization of endotoxin and suppression of TNF-a release in endotoxemic rats In our in vivo experiments, all rats were successfully fitted with cardiac catheters, showed no ill effects from the surgery, and no bacteria were found in blood cultures taken throughout the experiment. In rats challenged with sublethal LPS, we tested the plasma endotoxin and serum TNF-a level. Forty mg/kg

4. Discussion In the present study, biosensor technology was used to investigate the specific interactions of differ-

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Fig. 8. Survival of mice challenged by endotoxin. One hundred and twenty mice were randomly divided into six groups (n = 20). Groups of mice were treated with:
, LPS (20 mg/kg) in group 1; o, PGG alone (80 mg/kg) in group 2; 5, LPS (20 mg/kg) plus the PGG (20 mg/kg) in group 3; R , LPS (20 mg/kg) plus PGG (40 mg/kg) in group 4; 4, LPS (20 mg/kg) plus PGG (80 mg/kg) in group 5; E, LPS (20 mg/kg) plus PMB (1 mg/kg) in group 6. The total injection volume was 0.2 ml per 20 g bodyweight. The general conditions and mortality of the mice were observed for 7 days. Mice were fed ad libitum during the experiment. *p b 0.05 compared with LPS group; **p b 0.01 compared with LPS group. Other details are as described under Methods.

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PGG (mg/kg) Fig. 9. PGG reduced the plasma endotoxin levels in mice challenged with a sublethal LPS dose. Twenty mice were randomly divided into five groups (n = 4) that were intravenously injected as follows: LPS (50 Ag/kg) in group 1, LPS (50 Ag/kg) plus PGG (1 mg/kg) in group 2, LPS (50 Ag /kg) plus PGG (2 mg/kg) in group 3, LPS (50 Ag/kg) plus PGG (4 mg/kg) in group 4, and only saline in group 5 (control). The total injection volume was 0.2 ml per 20 g bodyweight. Mice were sacrificed 0.5 h after injection with LPS; blood samples were collected from the heart. Five microlitres of the plasma was diluted in 195 Al of saline. The endotoxin level in the diluent was assayed using the LAL test. *p b 0.05 compared with LPS; **p b 0.01 compared with LPS. Other details are as described under Methods.

ent components in traditional Chinese herbs with LPS, using a lipid A-coated surface. Optical biosensors are powerful tools for the analysis of biomolecular interactions, with the main advantage that no fluorescent or radiolabeled molecules are required [11]. Therefore, if there was an anti-endotoxin component in the herb extract, it should have specifically bound to the LPS/lipid. An immobilized on the hydrophobic surface of the biosensor, as monitored by changes in the refractive index in the sensor chip surface due to surface plasmon resonance. Generally, optical biosensors are mostly used to determine binding constants of ligands to receptors [12], here we show that it is also a very useful tool to screen for new components in natural products. In the present work, the aqueous extractions from 42 traditional Chinese herbs were tested, eight herbs showing lipid A-binding activities (Fig. 1). They are Belaminda Chineusis, Colla Corii Asini, Radix Paeoniae Rubras, Radix et Rhizoma Rhei, Fructus Aurantii Immaturus, Chinese White Olive, Fructus Gardeniae, and Aconitum Carmichaeli Deb. Among these eight herbs, Radix Paeoniae Rubras had the

greatest binding capacity, hence, an extract was investigated further. During the course of purification of the monomers, biosensor technology was also used to confirm whether there was a monomer with anti-endotoxin activity in the sample, which we would term as ddirectional separationT. After traditional methods were used to purify the components, we obtained 1, 2, 3, 4, 6-h-d-pentagalloyl-glucose (PGG), which we found possessed anti-endotoxin activity. Two other two monomers were also obtained, the structure of which remains to be confirmed. These results show that biosensor tech-

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Time (hour) Fig. 10. PGG reduced plasma endotoxin (A) and serum TNF-a (B) levels in rats challenged with a sublethal dose of LPS. Rats were randomly divided into two groups (n = 4) and treated with n, LPS (2 mg/kg); 5, PGG (40 mg/kg) and LPS (2 mg/kg). Blood samples (0.3 ml) were drawn at 0, 0.5, 1, 2, 4, 8, 12 and 24 h after inception of the experiment; the serum was stored at 80 8C for subsequent TNF-a assay. Meanwhile 10 Al of the plasma was diluted in 990 Al of saline twice, and the solution was then incubated in 90 8C water for 10 min in order to inactivate the heparin. The experiment was repeated twice. Other details are as described under Methods.

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nology is a useful method for obtaining antiendotoxin components from herbs. Importantly, it was found that the LAL assay, as a test for anti-endotoxin activity in aqueous extractions of herbs, was unreliable, and gave false positives, likely because there are many other compounds that interfere in the reaction. By contrast, biosensor technology does not suffer from interference from such compounds, and is therefore a better approach as compared with the LAL assay. PGG, being a tannic acid, was first isolated and purified from the freshwater green alga Spirogyra varians or a Paraguayan crude drug, bMolle-iQ (Schinus terebinthifolius) [13,14], and it has also been synthesized [15]. It is reported to be a xanthine oxidase inhibitor and to exert an effect on mitochondrial respiration and oxidative phosphorylation through action on the membrane and on succinate dehydrogenase, NADH dehydrogenase and on the cytochrome c complex of mitochondria [14,16]. In 1991, the water, water/acetone and methanol extracts of peony roots, paeoniflorin, albiflorin and pentagalloylglucose were studied for anticonvulsant action in rats. PGG was thought to be responsible for the anticonvulsant action [17]. In 1996, an extract prepared from the roots of Paeonia lactiflora relaxed prostaglandin F2a-precontracted aortic ring preparations of isolated rat aorta that contained endothelium [18]. In 2001, PGG was reported to inhibit LPS-stimulated TNF-a secretion in hPBMC and in rats [19]. However, the molecular mechanism of PGG on LPS effects has remained unclear. In our study, we first found PGG could significantly protect mice from an LPS challenge. Our in vitro experiments confirmed that PGG directly bound to lipid A with a Kd value of 3.2 AM. Also it could neutralize endotoxin, as assessed by both the LAL assay and in a TNF-a release experiment, in a dosedependent manner. The levels of endotoxin paralleled that of cytokines. Overall the results from our in vivo and in vitro experiments yielded concordant results. Taken together, our results show that affinity biosensor technology is a credible and effective method to screen for new anti-endotoxin agents from traditional Chinese herbs. PGG can significantly protect mice from an LPS challenge by decreasing plasma endotoxin and serum TNF-a release.

Acknowledgements This work was supported by a grant from the National Natural Science Foundation of China 30371767.

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