Tox(ron, Vol . 17, pp . 324-327. © Peraamon Press Ltd . 1979. Printed in Groat Britain .
0041-0101179/0901-0524502 .00/0
DETOXIFICATION BY PERSIMMON TANNIN OF SNAKE VENOMS AND BACTERIAL TOXINS TAKASHI OKONOGI, ZENPACHIRO HATTORI, AKIRA OGISO and SEIII 1VlITSUI Central Research Laboratories, Sankyo Co ., Ltd ., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo, 140, Japan (Accepted jor publication 23 January 1979)
to treat snake bite quickly and effectively, it is necessary to rapidly detoxify the snake venom. As indicated in this paper, tannin extracted from persimmon fruit is remarkably effective against snake venom as well as some toxic substances produced by microorganisms. Fresh unripe fruits (10 kg) of the oriental persimmon (Diospyros kaki Thunb .) having seeds and calyces removed were heated at 120°C for l hr in a small amount of distilled water. The fruit was then crushed and extracted with 151iters ofacetone at room temperature for 24 hr. The filtered acetone extract was concentrated to about 2 liters and to this concentrate 10 liters of methanol were added and the mixture was filtered again . To the filtrate were added 8 liters of diethyl ether to give a precipitate, which was separated and dissolved in 7~5 liters of distilled water, adjusted to pH 4 with hydrochloric acid, placed in a cellulose tube and dialysed in running water. The resulting dialysed solution was lyophilized to give 50 g of condensed tannin as a pale brown fine powder . The lyophilized tannin, [a] no x-90° (c = 1~0, water), showed absorption bands at 3400, 1615 and 1530 cm -1 in the infra-red spectrum and ultraviolet absorption at 275 nm. The persimmon tannin thus obtained was characterized to be a homogeneous and macromolecular condensed tannin having a proanthocyanidine structure from the following results . A chromatogram of a gel filtration using styrene-divinylbenzene sulphonate resin and water as a mobile phase, showed a single tannin peak, and an ultra-centrifuge Schlieren pattern measured on a 1 ~ solution in 0~ I M phosphate buffer (46,633 rev/min) proved to be homogeneous . The tannin was heated at 100°C in n-butanol containing 5 °,ô of conc. hydrochloric acid for 2 hr to give cyanidin and delphinidin showing a ratio of 1 :3. Furthermore, a sample of the condensed tannin was subjected to alkali fusion at 185°C under nitrogen for 10 min to give protocatechuic acid and gallic acid at a ratio of 1 :3 in addition to some phloroglucinol . The Lv su values of the persimmon tannin were 521 mg/kg i.v. (95 ~ confidence limits ; 4168-6513) and beyond 30 gJkg p.o. in mice when tested by the Litchfield-Wilcoxon method . The persimmon tannin in concentrations of less than I ~25 ~ (0~ I ml per mouse) caused hardly any swelling in the sole of the foot following injection, while 0625 ~ or above of tannic acid produced intense swelling . The effect of persimmon tannin was compared with that of other commercial tannins against Laticauda semifasciata Reinwardt (Erabu sea snake) venom (Table 1). Table 2 shows the effects of persimmon tannin and IN ORDER
524
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tannic acid against the venoms of Laticauda semifasciata and Trimeresurus flavoviridis Hallowell (Habu land snake) . Only 36 Fig of the persimmon tannin was sufficient to detoxify twice the 50 ~ lethal dose of Laticauda semifasciata venom in the mouse, while 1200 lIg oftannic acid was necessary to neutralize the same amount of the venom. In the case of Trimeresurus ,~lavoviridis venom, twice the minimal hemorrhagic dose of the venom was inactivated in the presence of 78 N~g of the persimmon tannin, while 5000 lIg tannic acid was required . TABLE 1 . DETOXIFYING EFFECT OF PERSIMMON TANNIN AND COMMERCIAL TANNINS AGAINST faSCtata VENOM
Samples Persimmon Quebracho Mimosa Myrabolam Chestnut Tannic acid
1000 5/5 3/5 (75) 0/5 (65) 0/5 (~ 5/5 5/5
500 5/5 0/5 (52) 0/5 (48) 0/5 (50) 0/5 (82) 2/5 (125)
Dose of tannin (lIg/mouse) 250 125 5/5 5/5 0/5 (47) 0/5 (47) 0/5 (37) 0/5 (35) 0/5 (46) 0/5 (26) 0/5 (70) 0/5 (68) 0/5 (86) 0/5 (56)
625 5/5 0/5 (35) 0/5 (30) 0/5 (30) 0/5 (47) 0/5 (56)
LatICalIlta
Sel>d-
3125 0/5 (50) 0/5 (31) 0/5 (32) 0/5 (29) 0/5 (40) 0/5 (33)
The mixture of tannin solution and 2 LDsn amounts of the venom were injected i.m . (0~2 ml) in mice. value of the venom was 637 hg/kg (95~ confidence limits ; 633-641) when tested by the LitchfieldWilcoxon method in mice. Numerators indicate the number of mice survived and denominators total number of mice injected. Average time of survival of mice in min are shown in parentheses. LD6,
TABLE
2.
COMPARL90N OF EFFICACY OF PERSIMMON TANNNN AND TANNIC ACID AGAINST SNAKB VENOMS
Snake venom Laticauda semifasciata venom : 2 LD6, 7}imeresurus ftavovtridts venom: 2 MIIn
Snake venom Laticauda semifasciata tannin : 5 mg Trtmeresurus,ftavovtrktis tannin : 5 mg
Tannin dilution Persimmon tannin Tannic acid ilg/0~1 ml IIg/0~1 ml Survival Survival 39 5/5 3/3 1250 19 0/5 0/3 625 Hemorrhage Hemorrhage 78 negative 5000 negative 39 positive 2500 positive Venom dilution Persimmon tannin Venom dose Survival 120 LD~o 5/5 180 LD,o 0/5 40 MI~m 600 MxD
Hemorrhage negative positive
Tannic acid Venom doso Survival 7~5 LD 5/5 10 LD,o
50 75
MIm MxD
0/S
Hemorrhage negative positive
In the case of tannin dilution experiments, tannin solutions were mixed with 2 LD bo of L. sernifasciata or 2 MILD of T.ffavovtrldis venom. In venom dilution experiments, 5 mg of tannin were mixed with solutions of the venom . These solutions then were injected i.m . (0~2 ml) in groups of 5 mice . Twenty-four hours after, survival and local hemorrhage were inspected. The minimal hemorrhaggc dose (Mrro) defined as that amount of venom resulting in a distinct hemorrhagc area 24 hr after injection. Mtln value of T. Jtavovlridis venom corresponds to 156 ug/mouse. Numerators indicate the number of mice survived and denominators total number of mice injected .
Binding of the persimmon tannin to the venom of Iaticauda semijasciata was so firm that the venom could not be separated by centrifugation, and no specific precipitate with the venom antiserum (OICONOGI et al., 1970) was observed on agar immunodiffusion plate by the OUCHTERLONY technique (Fig. 1). In contrast, snake venoms bound to tannic acid
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were separable by centrifugation and a precipitin with each snake venom antiserum emerged in the agar plate on standing over one week . These data suggest that the binding potency of the persimmon tannin to the venom proteins is strong and almost irreversible, while a hydrolysable tannin is bound loosely to the proteins and the resultant complex probably undergoes dissociation .
FIa. 1 . OUCHTERLONY DIFFUSION PLATE OF LrrtiCauda SenrljaSClata VENOM TO ANTIVENOM S : Trial manufacture of Laticauda semifasciata antivenin. V : L. semifasciata venom 2 mg/ml. V -I- P-Ex : L. senrljasciata venom 2 mgJml ~- 0~3% persimmon tannin (Mix . ratio ; 1 :1). V + T.A : L. semifasciata venom 2 mg/ml -f- 0~3 % tannic acid (Mix. ratio ; 1 :1).
Table 3 shows the potent detoxification effect of persimmon tannin against several bacterial toxins ; diphtheria, staphylococcus, tetanus and pertussis toxin. Persimmon tannin could also prevent in Bordetella pertussis vaccine the lymphocyte promoting factor as shown in Table 4. TABLE
3.
INACTIVATION OF BACTERIAL TOXINS
Bacterial toxins Corynebacterlum dlphtheriae exotoxin : 20 LD~o Staphylococcus aureus alpha toxin : 2 LD~u
Persimmon tanin (ug) 300 130 35 173
Clostridium tetani exotoxin :100 Ln, o
300 150
Bordetella pertussis Gundel toxin : 10 MAD
125 625
Survival (Guinea pig) S/S 0/S (mouse) S/S 0/S (mouse) S/S 0/S Hemorrhage (rabbit) negative positive
The toxin solutions were mixed with an equal volume of sequentially 2-fold diluted solutions of persimmon tannin. After standing for 10 min at room temperature, the resultant solution (0~2 ml) was applied as follows : diptheria toxin was injected s.c. to Hartley guinea pigs weighing 300 g, followed by observing fatality for 7 days, Staphylococcus alpha toxin was injected i.v . to DDY male mouse 25 f 1 g, followed by observing fatality for 3 days, pertussis Gundel toxin was intradermally injected in the dorsal skin of New Zealand white male rabbit, followed by observing hemorrhage in dorsal back skin after 24 hr . The LD yo values of the toxins were tested by Litchfield-Wilcoxon method ; Corynebacterium diphtheriae 889 ug/kg (93 % confidence limits ; 864-927) s.c. in guinea~pigs, Staphylococcus aureus 198 ug/kg (193-202) i.v . in mice, Clostridium tetani 356 ug/kg (334-380) s.c . in mice. The minimal hemorrhagic dose (MHD) defined as that amount of toxin resulting in a distinct hemorrhagic area 24 hr after injection. MHD value of pertussis Gundel toxin corresponds to 0625 lIg/rabbit . Numerators indicate the number of animals survived and denominators total number of animals injected .
Short Communications TABLE
4.
EFFECT OF PERSIMMON TANMN ON THE LYMPHOCYTE PROMOTING FACTOR OF PERTUSSIS VACCINE
Pertussis vaccine (Number of killed organisms) 1 x 101°/0~5 ml/mouse
Control
527
Persimmon tannin (ug/0~5 ml) 500 100 20 4 0
Number of leucocyte (cells/mm') 4860 f 445 6200 = 775 13,320 ~ 1154 26,620 f 1954 22,820 f 2334 4310 f 245
Pertussis vaccine solution (No. of killed organisms; 4 x 101°/ml) was mixed with an equal volume of sequentially 5-fold diluted persimmon tannin solutions in sterilized physiological saline . After standing at room temperature for 10 min, 0~5 ml of each treated vaccine solution was administered i.p . to DDY male mouse (n = 5) . As a control, the vaccine or a physiological saline were likewise injected . The number of mouse leucocyte was counted after 3 days .
The neutralizing effect of tannic acid against snake venom has been reported previously 1970, 1974 ; OxoxoGl, 1973 ; HOMMA et al., 1965). The persimmon tannin used in this experiment is stable to heat, readily soluble in water and of lower toxicity than tannic acid. The use of persimmon tannin is therefore more convenient in attempting inactivation of snake venoms or bacterial toxins. (OKONOGI et al .,
Acknowledgement-The authors are grateful to Professor I. KATO of the Institute of Medical Science, University of Tokyo, for his gifts of diphtheria and staphylococcus toxins . REFERENCES
Aee, R., OKONOGI, T., KOSUGE, T. and MISHIMA, S. (1965) Studies on Habu snake and Erabu sea snake venoms . Outlines of biological toxicities of two snake venoms and inhibitory actions of tannic acid on them . Jap. J. Bact . 20, 281. OKONOGI, T. (1973) Venomous sea snake bite . The Snake 5, 156. OICONOGI, T., HATTORI, Z. SIId AMAGI, E. (1970) The emergency treatment of poisonous snake bite with tannic acid solution . The Snake 2, 106. OKONOGI, T., HATTORI, Z., WATANAHE, M. and AMAGI, E. (1970) Neutralization of sea snake venom with goat antivenin. The Snake 2, 18 . HOMMA, M.,
0041-0101179/0901-0524502 .00/0
DETOXIFICATION BY PERSIMMON TANNIN OF SNAKE VENOMS AND BACTERIAL TOXINS TAKASHI OKONOGI, ZENPACHIRO HATTORI, AKIRA OGISO and SEIII 1VlITSUI Central Research Laboratories, Sankyo Co ., Ltd ., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo, 140, Japan (Accepted jor publication 23 January 1979)
to treat snake bite quickly and effectively, it is necessary to rapidly detoxify the snake venom. As indicated in this paper, tannin extracted from persimmon fruit is remarkably effective against snake venom as well as some toxic substances produced by microorganisms. Fresh unripe fruits (10 kg) of the oriental persimmon (Diospyros kaki Thunb .) having seeds and calyces removed were heated at 120°C for l hr in a small amount of distilled water. The fruit was then crushed and extracted with 151iters ofacetone at room temperature for 24 hr. The filtered acetone extract was concentrated to about 2 liters and to this concentrate 10 liters of methanol were added and the mixture was filtered again . To the filtrate were added 8 liters of diethyl ether to give a precipitate, which was separated and dissolved in 7~5 liters of distilled water, adjusted to pH 4 with hydrochloric acid, placed in a cellulose tube and dialysed in running water. The resulting dialysed solution was lyophilized to give 50 g of condensed tannin as a pale brown fine powder . The lyophilized tannin, [a] no x-90° (c = 1~0, water), showed absorption bands at 3400, 1615 and 1530 cm -1 in the infra-red spectrum and ultraviolet absorption at 275 nm. The persimmon tannin thus obtained was characterized to be a homogeneous and macromolecular condensed tannin having a proanthocyanidine structure from the following results . A chromatogram of a gel filtration using styrene-divinylbenzene sulphonate resin and water as a mobile phase, showed a single tannin peak, and an ultra-centrifuge Schlieren pattern measured on a 1 ~ solution in 0~ I M phosphate buffer (46,633 rev/min) proved to be homogeneous . The tannin was heated at 100°C in n-butanol containing 5 °,ô of conc. hydrochloric acid for 2 hr to give cyanidin and delphinidin showing a ratio of 1 :3. Furthermore, a sample of the condensed tannin was subjected to alkali fusion at 185°C under nitrogen for 10 min to give protocatechuic acid and gallic acid at a ratio of 1 :3 in addition to some phloroglucinol . The Lv su values of the persimmon tannin were 521 mg/kg i.v. (95 ~ confidence limits ; 4168-6513) and beyond 30 gJkg p.o. in mice when tested by the Litchfield-Wilcoxon method . The persimmon tannin in concentrations of less than I ~25 ~ (0~ I ml per mouse) caused hardly any swelling in the sole of the foot following injection, while 0625 ~ or above of tannic acid produced intense swelling . The effect of persimmon tannin was compared with that of other commercial tannins against Laticauda semifasciata Reinwardt (Erabu sea snake) venom (Table 1). Table 2 shows the effects of persimmon tannin and IN ORDER
524
52 5
Short Communications
tannic acid against the venoms of Laticauda semifasciata and Trimeresurus flavoviridis Hallowell (Habu land snake) . Only 36 Fig of the persimmon tannin was sufficient to detoxify twice the 50 ~ lethal dose of Laticauda semifasciata venom in the mouse, while 1200 lIg oftannic acid was necessary to neutralize the same amount of the venom. In the case of Trimeresurus ,~lavoviridis venom, twice the minimal hemorrhagic dose of the venom was inactivated in the presence of 78 N~g of the persimmon tannin, while 5000 lIg tannic acid was required . TABLE 1 . DETOXIFYING EFFECT OF PERSIMMON TANNIN AND COMMERCIAL TANNINS AGAINST faSCtata VENOM
Samples Persimmon Quebracho Mimosa Myrabolam Chestnut Tannic acid
1000 5/5 3/5 (75) 0/5 (65) 0/5 (~ 5/5 5/5
500 5/5 0/5 (52) 0/5 (48) 0/5 (50) 0/5 (82) 2/5 (125)
Dose of tannin (lIg/mouse) 250 125 5/5 5/5 0/5 (47) 0/5 (47) 0/5 (37) 0/5 (35) 0/5 (46) 0/5 (26) 0/5 (70) 0/5 (68) 0/5 (86) 0/5 (56)
625 5/5 0/5 (35) 0/5 (30) 0/5 (30) 0/5 (47) 0/5 (56)
LatICalIlta
Sel>d-
3125 0/5 (50) 0/5 (31) 0/5 (32) 0/5 (29) 0/5 (40) 0/5 (33)
The mixture of tannin solution and 2 LDsn amounts of the venom were injected i.m . (0~2 ml) in mice. value of the venom was 637 hg/kg (95~ confidence limits ; 633-641) when tested by the LitchfieldWilcoxon method in mice. Numerators indicate the number of mice survived and denominators total number of mice injected. Average time of survival of mice in min are shown in parentheses. LD6,
TABLE
2.
COMPARL90N OF EFFICACY OF PERSIMMON TANNNN AND TANNIC ACID AGAINST SNAKB VENOMS
Snake venom Laticauda semifasciata venom : 2 LD6, 7}imeresurus ftavovtridts venom: 2 MIIn
Snake venom Laticauda semifasciata tannin : 5 mg Trtmeresurus,ftavovtrktis tannin : 5 mg
Tannin dilution Persimmon tannin Tannic acid ilg/0~1 ml IIg/0~1 ml Survival Survival 39 5/5 3/3 1250 19 0/5 0/3 625 Hemorrhage Hemorrhage 78 negative 5000 negative 39 positive 2500 positive Venom dilution Persimmon tannin Venom dose Survival 120 LD~o 5/5 180 LD,o 0/5 40 MI~m 600 MxD
Hemorrhage negative positive
Tannic acid Venom doso Survival 7~5 LD 5/5 10 LD,o
50 75
MIm MxD
0/S
Hemorrhage negative positive
In the case of tannin dilution experiments, tannin solutions were mixed with 2 LD bo of L. sernifasciata or 2 MILD of T.ffavovtrldis venom. In venom dilution experiments, 5 mg of tannin were mixed with solutions of the venom . These solutions then were injected i.m . (0~2 ml) in groups of 5 mice . Twenty-four hours after, survival and local hemorrhage were inspected. The minimal hemorrhaggc dose (Mrro) defined as that amount of venom resulting in a distinct hemorrhagc area 24 hr after injection. Mtln value of T. Jtavovlridis venom corresponds to 156 ug/mouse. Numerators indicate the number of mice survived and denominators total number of mice injected .
Binding of the persimmon tannin to the venom of Iaticauda semijasciata was so firm that the venom could not be separated by centrifugation, and no specific precipitate with the venom antiserum (OICONOGI et al., 1970) was observed on agar immunodiffusion plate by the OUCHTERLONY technique (Fig. 1). In contrast, snake venoms bound to tannic acid
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Short Communications
were separable by centrifugation and a precipitin with each snake venom antiserum emerged in the agar plate on standing over one week . These data suggest that the binding potency of the persimmon tannin to the venom proteins is strong and almost irreversible, while a hydrolysable tannin is bound loosely to the proteins and the resultant complex probably undergoes dissociation .
FIa. 1 . OUCHTERLONY DIFFUSION PLATE OF LrrtiCauda SenrljaSClata VENOM TO ANTIVENOM S : Trial manufacture of Laticauda semifasciata antivenin. V : L. semifasciata venom 2 mg/ml. V -I- P-Ex : L. senrljasciata venom 2 mgJml ~- 0~3% persimmon tannin (Mix . ratio ; 1 :1). V + T.A : L. semifasciata venom 2 mg/ml -f- 0~3 % tannic acid (Mix. ratio ; 1 :1).
Table 3 shows the potent detoxification effect of persimmon tannin against several bacterial toxins ; diphtheria, staphylococcus, tetanus and pertussis toxin. Persimmon tannin could also prevent in Bordetella pertussis vaccine the lymphocyte promoting factor as shown in Table 4. TABLE
3.
INACTIVATION OF BACTERIAL TOXINS
Bacterial toxins Corynebacterlum dlphtheriae exotoxin : 20 LD~o Staphylococcus aureus alpha toxin : 2 LD~u
Persimmon tanin (ug) 300 130 35 173
Clostridium tetani exotoxin :100 Ln, o
300 150
Bordetella pertussis Gundel toxin : 10 MAD
125 625
Survival (Guinea pig) S/S 0/S (mouse) S/S 0/S (mouse) S/S 0/S Hemorrhage (rabbit) negative positive
The toxin solutions were mixed with an equal volume of sequentially 2-fold diluted solutions of persimmon tannin. After standing for 10 min at room temperature, the resultant solution (0~2 ml) was applied as follows : diptheria toxin was injected s.c. to Hartley guinea pigs weighing 300 g, followed by observing fatality for 7 days, Staphylococcus alpha toxin was injected i.v . to DDY male mouse 25 f 1 g, followed by observing fatality for 3 days, pertussis Gundel toxin was intradermally injected in the dorsal skin of New Zealand white male rabbit, followed by observing hemorrhage in dorsal back skin after 24 hr . The LD yo values of the toxins were tested by Litchfield-Wilcoxon method ; Corynebacterium diphtheriae 889 ug/kg (93 % confidence limits ; 864-927) s.c. in guinea~pigs, Staphylococcus aureus 198 ug/kg (193-202) i.v . in mice, Clostridium tetani 356 ug/kg (334-380) s.c . in mice. The minimal hemorrhagic dose (MHD) defined as that amount of toxin resulting in a distinct hemorrhagic area 24 hr after injection. MHD value of pertussis Gundel toxin corresponds to 0625 lIg/rabbit . Numerators indicate the number of animals survived and denominators total number of animals injected .
Short Communications TABLE
4.
EFFECT OF PERSIMMON TANMN ON THE LYMPHOCYTE PROMOTING FACTOR OF PERTUSSIS VACCINE
Pertussis vaccine (Number of killed organisms) 1 x 101°/0~5 ml/mouse
Control
527
Persimmon tannin (ug/0~5 ml) 500 100 20 4 0
Number of leucocyte (cells/mm') 4860 f 445 6200 = 775 13,320 ~ 1154 26,620 f 1954 22,820 f 2334 4310 f 245
Pertussis vaccine solution (No. of killed organisms; 4 x 101°/ml) was mixed with an equal volume of sequentially 5-fold diluted persimmon tannin solutions in sterilized physiological saline . After standing at room temperature for 10 min, 0~5 ml of each treated vaccine solution was administered i.p . to DDY male mouse (n = 5) . As a control, the vaccine or a physiological saline were likewise injected . The number of mouse leucocyte was counted after 3 days .
The neutralizing effect of tannic acid against snake venom has been reported previously 1970, 1974 ; OxoxoGl, 1973 ; HOMMA et al., 1965). The persimmon tannin used in this experiment is stable to heat, readily soluble in water and of lower toxicity than tannic acid. The use of persimmon tannin is therefore more convenient in attempting inactivation of snake venoms or bacterial toxins. (OKONOGI et al .,
Acknowledgement-The authors are grateful to Professor I. KATO of the Institute of Medical Science, University of Tokyo, for his gifts of diphtheria and staphylococcus toxins . REFERENCES
Aee, R., OKONOGI, T., KOSUGE, T. and MISHIMA, S. (1965) Studies on Habu snake and Erabu sea snake venoms . Outlines of biological toxicities of two snake venoms and inhibitory actions of tannic acid on them . Jap. J. Bact . 20, 281. OKONOGI, T. (1973) Venomous sea snake bite . The Snake 5, 156. OICONOGI, T., HATTORI, Z. SIId AMAGI, E. (1970) The emergency treatment of poisonous snake bite with tannic acid solution . The Snake 2, 106. OKONOGI, T., HATTORI, Z., WATANAHE, M. and AMAGI, E. (1970) Neutralization of sea snake venom with goat antivenin. The Snake 2, 18 . HOMMA, M.,
