The Journal of Toxicological Sciences, 441 Vol.27, No.5, 441-447, 2002

A MECHANISM OF THE FRACTION OF (CAMELLIA SINENSIS) EXTRACT PROTECTING AGAINST THE EFFECT OF TETANUS TOXIN

Eiki SATOH1, Toshiaki ISHII1, Yoshio SHIMIZU2, Shin-ichi SAWAMURA3 and Masakazu NISHIMURA1

1Department of Pathobiological Science, Obihiro University of Agriculture and Veterinary Medicine, 11, West-2, Inada, Obihiro 080-8555, Japan 2Cooperative Research Center, Obihiro University of Agriculture and Veterinary Medicine, 11, West-2, Inada, Obihiro 080-8555, Japan 3Ito En, Ltd., 3-47-10, Honmachi, Shibuya Ward, Tokyo 151-0071, Japan

(Received June 10, 2002; Accepted August 26, 2002)

ABSTRACT — The aim of the present study was to elucidate the mechanism of the protective effect of black tea extract’s thearubigin fraction against the action of tetanus toxin. The effects of thearubigin frac- tion extracted from a black tea infusion were examined for neuromuscular blocking action on tetanus toxin in mouse phrenic nerve-diaphragm preparations and on the binding of this toxin to the synaptosomal mem- brane preparations of rat cerebral cortices. The interaction between tetanus toxin and thearubigin fraction was also investigated. Tetanus toxin (4 µg/ml) abolished indirect twitches in mouse phrenic nerve-dia- phragm preparations within 150 min. Thearubigin fraction mixed with tetanus toxin blocked the inhibitory effect of the toxin. Mixing iodinated toxin with thearubigin fraction inhibited the specific binding of [125I]tetanus toxin to the synaptosomal membrane preparation. The effects of thearubigin fraction were dose-dependent. The elution profile of [125I]tetanus toxin on Sephadex G-50 column chromatography was different from that of toxin mixed with thearubigin fraction. These findings indicate that thearubigin frac- tion protects against the action of tetanus toxin by binding with the toxin.

KEY WORDS: Tetanus toxin, Thearubigin fraction, Phrenic-nerve diaphragm, Synaptosomal membrane, Specific binding, Black tea

INTRODUCTION (Habermann et al., 1980). Botulinum neurotoxin pro- duced by C. botulinum, another clostridial neurotoxin, Tetanus toxin produced by Clostridium tetani has a similar action and causes botulism in human (Pel- exhibits the strongest neurotoxicity and has caused tet- lizzari et al., 1999). These neurotoxins are composed of anus in mammals. The toxin invading from a wound protein. The recent interest in clostridial neurotoxins binds to the presynaptic membrane of the neuromuscu- comes from the potential threat that they might be used lar junction, and is then internalized and transported by terrorist groups or other nations as biological weap- retroaxonally to the spinal cord. The spastic paralysis ons (Henderson, 1999). induced by the toxin is due to the blockade of neu- We have continued an effort to find an inactivator rotransmitter release from spinal inhibitory neurons for clostridial neurotoxins in teas (black tea, oolong tea, (Pellizzari et al., 1999). On the other hand, tetanus roasted tea and green tea) and observed that black tea toxin blocks neuromuscular transmission by inhibiting infusion, oolong tea infusion and roasted tea infusion the release of neurotransmitters from motor nerve ter- protected against toxicity from tetanus toxin in mouse minals and thereby causes flaccid paralysis phrenic nerve-diaphragm preparations (Satoh et al.,

Correspondence: Masakazu NISHIMURA

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2001b). The potency order of the protecting effects al. (1993). After the extraction of the caffeine fraction was: black tea infusion > oolong tea infusion > roasted (chloroform extract), and the and frac- tea infusion. Green tea infusion and some components tion (ethyl acetate extract), the tea infusion sample (40 ml) in tea infusion (e.g., tannic acid, catechin, and caffeine) was extracted with 40 ml of 1-butanol. The butanol had no effects. For the manufacture of black tea, the extract was dried with a rotatory evaporator in a vacuum. fermentation process causes green tea polyphenols to The residue (240 mg) was dissolved in 3 ml of distilled oxidize and form oligomeric flavalols, including water for preparation of the thearubigin fraction. Total , and other oligomers phenolic content (polyphenols, % weight/extract weight) (Hazarika et al., 1984; Roberts, 1958). Theaflavins and in the thearubigin fraction was greater than 80% as mea- thearubigins are contained in the infusion of black tea sured according to the method described by Singleton and (fermented tea) and oolong tea (semifermented tea), Rossi (1965). although the content in the infusion of oolong tea is lower than that of black tea (Xie et al., 1993). The infu- In vitro studies sion of green tea contains a very small amount of The phrenic nerve-diaphragm preparations were theaflavins, but not thearubigins. Thearubigins are made from ddY strain male and female mice (30-35 g). complex polyphenols of poorly-defined chemical The preparation was soaked in a modified Krebs- structures. The thearubigin fraction protects against Ringer solution of the following composition (in mM): subcutaneous toxicity by tetanus toxin in mice, NaCl 136; KCl 5; CaCl2 2; MgCl2 1; NaHCO3 15; glu- although theaflavins failed (Satoh et al., 2001b). The cose 11. The solution was bubbled with a mixture of protective effect of thearubigin fraction might be due to 95% O2 and 5% CO2 and maintained at a pH of 7.3 at binding of thearubigin fraction with the toxin, because 36°C. A basal loading tension of 1.0 g was applied to the protective effect occurred only by mixing thearubi- the preparation. The nerve trunk or muscle layer of the gin fraction with the toxin, but not with early or late diaphragm preparation soaked in the Krebs-Ringer administration of thearubigin fraction. The mechanism solution was stimulated with supramaximal square by which thearubigin fraction prevents the paralysis wave pulses at 0.1 Hz with an electronic stimulator induced by this toxin, however, is obscure. To elucidate (SEN 3301; Nihon Kohden, Tokyo, Japan). Isometric the precise mechanism of action of thearubigin frac- force development was recorded on a thermal array tion, we examined the effect of thearubigin fraction on recorder (AD 100F; Nihon Kohden). Because the the binding of tetanus toxin to the synaptosomal mem- observation period was prolonged, without exchanging brane preparations of rat cerebral cortices in addition the medium in the organ bath, tetanus toxin-induced to its effect on paralysis resulting from the toxin in paralysis was measured as a 20% reduction in the mus- mouse phrenic nerve-diaphragm preparations. Further- cle twitch response to nerve stimulation. more, we investigated the interaction between thearub- igin fraction and tetanus toxin. Binding studies Synaptosomal preparations (P2B fraction) were MATERIALS AND METHODS prepared using the method described by Hajós (1975) from the cerebral cortices of male and female Wistar Tea leaves and toxins rats (200-300 g). The synaptosomal preparations were Black tea (Camellia Sinensis) leaves were suspended in iced Tris-HCl buffer (50 mM, pH 7.4) obtained from the Cooperative Society (Tokyo, Japan). and frozen at −80°C until use. The thawed synaptoso- The tetanus toxin (Molecular Weight ≈150000) was mal preparations were suspended in iced Tris-HCl purchased from List Biological Laboratories (Camp- buffer,incubatedonicefor60min,andthencentri- bell, CA) and was dissolved in 0.01 M sodium phos- fuged (1000 × g) for 10 min at 4°C. The resulting phate buffer (pH 7.5). supernatant was centrifuged (40000 × g) for 45 min at 4°C. The pellet was washed in iced Tris-HCl buffer by Thearubigin fraction centrifugation (as described above) two times to pre- Black tea leaves (12 g) were incubated for 2 min pare synaptosomal membrane preparations. with 90 ml of hot distilled water (100°C). The tea infusion Tetanus toxin was iodinated according to the sample was filtered with filter paper (No. 2; Whatman, method described by Simpson et al. (1993). Toxin (150 Maidstone, England). A sample of thearubigin fraction µg) in sodium borate buffer (100 mM, pH 7.9) was from the tea infusion was prepared as described by Xie et mixed with [125I]Na (1 mCi; New England Nuclear,

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Boston, MA, USA) at room temperature. The reaction Statistical analysis wasterminatedafter30minbyaddingglycine(200 The statistical significance of differences was mM). [125I]Tetanus toxin was separated from reactants assessed using the Student’s t-test (Steel and Torrie, on a Sephadex G-50 column (Amersham Pharmacia 1980). A two-tailed p-value of less than 0.05 was con- Biotech, Uppsala, Sweden). Residual toxicity of the sidered to be statistically significant. iodinated preparation was bioassayed using the method of Kondo et al. (1984) and was 65% to 85%. RESULTS [125I]Tetanus toxin (0.3 nM) was mixed with a synaptosomal membrane preparation (50 µg protein) Effect of thearubigin fraction on toxin-induced in 1 ml of pH7.4 buffer containing 50 mM Tris-HCl, neuromuscular blockade 100 mM sodium chloride, and 1 mg/ml bovine serum Muscle twitches were elicited neurally (indirect albumin(Sigma,St.Louis,MO,USA).Thebinding twitches) and directly (direct twitches). Constant reaction was performed at 22°C for 30 min (Simpson et amplitude was maintained for at least 180 min. The al., 1993). The reaction was terminated by rapid filtra- amplitude of the indirect twitches was constant in the tion in a vacuum through a glass fiber-filter presence or absence of thearubigin fraction (80-800 (Whatman). The filter was then immediately washed µg/1-10 µl), showing neither an inhibitory nor an exci- three times with 5 ml of ice-cold binding buffer. Radio- tatory effect on the amplitude of the indirect twitches activity remaining on the filter was measured directly (data not shown). The indirect twitches were abolished with a gamma counter (Li and Singh, 1999). Specific by tetanus toxin (4 µg/ml, 80 µg/320 µl of tetanus binding to the synaptosomal membrane preparations toxin sample per 20 ml of organ bath medium) within was determined in parallel incubations containing a 150 min after exposure to the toxin (Fig. 2). The direct 500-fold excess of unlabeled tetanus toxin over labeled twitches were not affected by tetanus toxin for at least toxin. The protein levels of synaptosomal membrane 180 min (data not shown). Mixing tetanus toxin with preparations and tetanus toxin were quantified using a thearubigin fraction (160-640 µg/2-8 µl) prolonged the kit from Bio-Rad (Richmond, CA, USA), as described time to onset of paralysis induced by the toxin. The by Bradford (1976). prolonging effect was dependent on the dose of thearu- [125I]Tetanus toxin (50 µg/ml), thearubigin frac- bigin fraction (Fig. 1). The paralysis induced by teta- tion (8 mg/ml) or a mixture ([125I]tetanus toxin 50 µg/ nus toxin was inhibited by mixing the toxin with 500 µl; thearubigin fraction 8 mg/500 µl) was applied thearubigin fraction (160-800 µg/2-10 µl). The inhibi- to a Sephadex G-50 column (1.5 × 9 cm). The column tory effect was also dependent on the dose of thearubi- was eluted with pH 7.4 buffer (50 mM Tris-HCl, 100 gin fraction (Fig. 2). The early or late addition of mM sodium chloride, and 15% acetonitrile) to elute thearubigin fraction into the bath medium did not pro- tetanus toxin and thearubigin fraction. Fifty 1-ml frac- long the time to onset of paralysis induced by tetanus tions were collected and radioactivity in each fraction toxin and did not inhibit the paralytic effect of the toxin measured directly with a gamma counter. The ratios of (Fig. 1 and 2). Thearubigin fraction did not protect recovery of tetanus toxin and polyphenols (total phe- against other toxins such as tetrodotoxin and saxitoxin nolic content) were about 90% and 80%, respectively. (data not shown). The protective effect of thearubigin fraction was not modified by mixing thearubigin frac- Experimental animals tion (800 µg/10 µl) with bovine serum albumin (100 All procedures for the care and use of experimen- µg/10 µl) before mixing with toxin (data not shown). tal animals were approved by the Animal Research Committee, Obihiro University, and they were con- Effect of thearubigin fraction on toxin binding ducted under both the Guidelines for Animal Experi- To elucidate the mechanism underlying the pro- ments in Obihiro University and the Guiding Principles tective effect of thearubigin fraction against the effect in the Use of Animals in Toxicology that were adopted of tetanus toxin, the effect of thearubigin fraction on by the Society of Toxicology in 1989. the specific binding of labeled toxin to the presynaptic membrane was examined using the synaptosomal Chemicals membrane preparations of rat cerebral cortices. The All chemicals were purchased from Wako Pure specific binding of [125I]tetanus toxin to the synaptoso- Chemical Industries, Ltd. unless otherwise stated, and mal membrane preparations was inhibited by mixing were of 98.0% purity or greater. labeled toxin (45 ng/10 µl) with thearubigin fraction

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(4-32 µg/6.4-20 µl). This inhibitory effect was depen- ing labeled toxin (data not shown). Mixing [125I]tetanus dent on the dose of thearubigin fraction (Table 1). The toxin with thearubigin fraction shifted a peak to frac- present results were almost identical with our previous tion 19 in which polyphenols are rich (Fig.3). The elu- results (Satoh et al., 2001b). The early or late addition tion profile of polyphenols in the thearubigin fraction of thearubigin fraction to the synaptosomal suspension was identical regardless of thearubigin fraction only or failed to inhibit the specific binding of [125I]tetanus by mixing this fraction with the toxin (data not shown). toxin (Table 1). The inhibitory effect of thearubigin The elution profile of [125I]tetanus toxin was not modi- fraction was not modified by mixing thearubigin frac- fied by mixing thearubigin fraction (8 mg/500 µl) with tion (64 µg/32 µl) with bovine serum albumin (100 ng/ bovine serum albumin (50 µg/5 µl) before mixing with 2 µl) before mixing with toxin (data not shown). toxin (50 µg/500 µl) (data not shown). These results indicate that thearubigin fraction binds with tetanus Interaction between toxin and thearubigin fraction toxin and that its binding is specific. To clarify whether thearubigin fraction binds with tetanus toxin and its binding specifically, the DISCUSSION interaction between toxin and thearubigin fraction was investigated using Sephadex G-50 column. The chro- The present study demonstrated that thearubigin matogram of [125I]tetanus toxin on Sephadex G-50 col- fraction inhibits the specific binding of [125I]tetanus toxin umn showed a peak at fraction 12 (Fig.3). The elution to the synaptosomal membrane preparations of rat cere- profile was identical with that of an experiment isolat- bral cortices only when mixed together. Furthermore, it

Fig. 1. Effect of the thearubigin fraction on the rate of onset of paralysis caused by tetanus toxin in mouse phrenic nerve-diaphragm prepa- rations. A mixture of thearubigin fraction (80-640 µg/1-8 µl) with tetanus toxin (80 µg/320 µl) was added into Krebs-Ringer solution (20 ml). Tetanus toxin-induced paralysis was measured as a 20% reduction in muscle twitch response to nerve stimulation. Control (Cont) indicates tetanus toxin-induced response without mixing with thearubigin fraction. Each bar represents the mean ± S.E. of values from male (n = 3) and female (n = 3) mice. 1), 2): Thearubi- gin fraction was added into Krebs-Ringer solution before or after the addition of tetanus toxin. Values significantly different from the corresponding control level are indicated: *p <0.05, **p <0.01.

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Counteraction of tetanus toxin by thearubigin fraction. has been indicated that thearubigin fraction binds with tet- chain and a light chain that are linked by a disulfide anus toxin and its binding is specific. Thearubigin fraction bond. The carboxyl-terminal part of the heavy chain is thus inhibits the entry of tetanus toxin into motor nerve mainly responsible for the neuro-specific binding terminals by binding with the toxin and thereby blocking (Montecucco and Schiavo, 1995; Pellizzari et al., the paralytic effect induced by the toxin. 1999). In addition to tetanus toxin, we demonstrated Thearubigin fraction counteracts the paralytic that thearubigin fraction inhibits the entry of the botu- effect of tetanus toxin in a dose-dependent manner in linum neurotoxin types A, B, and E into nerve terminal mouse phrenic nerve-diaphragm preparations. This membranes by binding with the toxin, thus blocking protective effect of thearubigin fraction occurs only the paralytic effect of botulinum neurotoxins in vitro when it is mixed with tetanus toxin. This will be the and in vivo (Satoh et al., 2001a, 2002). Therefore, it is case with in vivo experiments as well (Satoh et al., suggested that thearubigin fraction affects the car- 2001b). These findings support the proposal that boxyl-terminal part of clostridial neurotoxins. thearubigin fraction blocks the paralytic effect of teta- The action of clostridial neurotoxins such as botuli- nus toxin by inhibiting the entry of the toxin into motor num neurotoxins and tetanus toxin involves three steps: nerve terminals through binding with toxin. extracellular binding and internalization, membrane Clostridial neurotoxins such as botulinum neuro- translocation, and proteolysis for specific components of toxins and tetanus toxin are strikingly similar in their the neuroexocytosis apparatus. There are three classes of macrostructures and are basically composed of a heavy universal antagonists that delay or abolish the actions of

Fig. 2. Effect of the thearubigin fraction on the paralysis caused by tetanus toxin in mouse phrenic nerve-diaphragm preparations. A mixture of thearubigin fraction (80-800 µg/1-10 µl) with tetanus toxin (80 µg/320 µl) was added into Krebs-Ringer solution (20 ml). Twitch tension (% initial twitch tension) indicates muscle twitch response to nerve stimulation 150 min after the application of tetanus toxin. Control (Cont) indicates tetanus toxin-induced response without mixing with thearubigin fraction. Each bar represents the mean ± S.E. of values from male (n = 3) and female (n = 3) mice. 1), 2): Thearubigin fraction was added into Krebs-Ringer solution before or after the addition of tetanus toxin.

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Table 1. Effect of the thearubigin fraction on the spe- all serotypes of botulinum neurotoxin and tetanus toxin: cific binding of [125I]tetanus toxin to the synap- 1) lectins with an affinity for sialic acid antagonize bind- tosomal membrane preparations. ing, 2) drugs that block (e.g., bafilomycin) or reverse (e.g., methylamine hydrochloride) acidification of endosomes µ 125 µ Thearubigin fraction ( g) [ I]Tetanus toxin bound (cpm/ g) antagonize internalization, and 3) drugs that chelate zinc ± 0 81.2 10.2 antagonize intracellular expression of toxicity (Simpson ± 1 73.1 8.6 et al., 1993). The mode of action of thearubigin fraction is ± 2 57.4 6.2 different from above three classes of antagonists. Namely, ± 4 38.8 7.3* thearubigin fraction directly binds with botulinum neuro- ± 8 14.3 4.0** toxins or tetanus toxin and blocks their extracellular bind- ± 16 6.2 1.5** ing. Thearubigin fraction has emerged as a universal ± 32 0.2 0.1*** inactivator of clostridial neurotoxins and is the first sub- 1) ± 32 78.5 9.7 stance to be identified from a natural foodstuff (black tea) 2) ± 32 75.6 12.9 as a broad spectrum inactivator of clostridial neurotoxins. Synaptosomal membrane preparations were prepared from the cerebral cortices of male (n = 3) and female (n = 3) rats. As the thearubigin fraction used in the present A mixture of thearubigin fraction (1-32 µg) and tetanus study is a crude preparation, it is necessary to isolate toxin (0.3 nM), or toxin alone was incubated with synapto- the effective substance(s) from the fraction. An early somal membrane preparation (50 mg/assay per ml) for 30 addition of concentrated active substance(s) must ° ± min at 22 C. Values represent the mean S.E. of six experi- inhibit the effects of tetanus toxin. Further experiments ments performed twice. 1), 2): Thearubigin fraction was added into the synaptosomal remain to elucidate the structure and character of the suspension before or after the addition of tetanus toxin. effective substance(s) in thearubigin fraction. *, **, ***: Significant difference from control (i.e., toxin only) (p<0.05, p<0.01, p<0.001). ACKNOWLEDGMENT

This work was supported by the Hokkaido Foun- dation for the Promotion of Scientific and Industrial

Fig. 3. Chromatograms of [125I]tetanus toxin ([125I]TeTx) and thearubigin fraction (TRB) on Sephadex G-50 column. : [125I]TeTx (50 µg/ ml), : A mixture of [125I]TeTx (50 µg/500 µl) and TRB (8 mg/ 500 µl), : TRB (8 mg/ml), Solvent: 15% (v/v) acetonitrile in Tris-HCl buffer (50 mM, pH 7.4, containing 100 mM sodium chlo- ride).

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