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Seasonal variability in the contents and composition of essential oil in various Thuja species occuring in Czechoslovakia
573
itisnof rat liver
cytosofk
ione
Merlos, M., Sgnchez, R.M., Camarasa, J. and Adze& T. Lubotatqv of Pharmacology and Pharmacognosy. School oj Pharmacy, University of Barcelona, Zona Univetsitatia de Pedralbes s/n, 08028 Barcelona, Spain
Glutathione S-transferases (GST) are a family of cytosolic multifunctional enzymes with an important role in detoxification processes. They catalyse the conjugation of electrophilic xenobiotics with the sulfhydryl moiety of glutathione (GSH) (Kaplowitz, 1980). In this study the “in vitro” inhibitory potencies of different plant flavonoids on rat liver cytosolic GST activity were compared. The GST activity was assayed according to Habig et al. (1974) by using 1-chloro-2&dinitrobenzene (CDNB) as substrate. The IC,, values varied over a 30-fold range. In a general manner, the order of potency found was flavonols > flavones > flavanonols. Flavone nucleus alone provided only mild inhibition (IC, = 0.93 mM). Activity was shown to depend on 1) the pattern of hydroxylation: Thus, the presence of both hydroxyl groups in 3’,4’ provided enhanced activity in flavonols and flavones (IC, = 31 and 42 PM for quercetin and luteolin, respectively). 4’-hydroxylated analogous were less potent (IC, = 76 and > 200 FM for kaempferol and apigenin). 7-substitution by a sugar moiety decreases activity (7-glucosyl-luteolin, IC, = 210 @I). Additional 2’-hydroxylation of morin increased activity slightly (I& = 41 PM) in comparison with kaempferol. 2) the presence of a double bond in C-2,C-3: A 20-fold loss of activity was obtained with the flavanonol taxifolin compared with its analogous flavonol quercetin. Therefore, the planarity of benzopyrone ring seems to be an important requirement for inhibition. The kinetics of inhibition of quercetin, the strongest inhibitor found, revealed a non competitive profile versus both GSH and CDNB, with apparent Hi values of 75 and 60 FM, respectively. Querc&n was shown to inhibit GSH conjugation with several other electrophilic substrates, such as 1,2-dichloro4-nitrobenzene and p-nitrobenzyl chloride. On the contrary, an activation of GSH - 1,2-epoxy-3-(p-nitrophenoxy)propane conjugation was noted. A selective interaction with one or several of the isoenzymes which possess GST activity is proposed. eferences N. Kaplowitz, 1980, Physiological significance of glutathione S-transferases. J. Physioi, 239, G439. Habig, W.H.. Pabst, M.J. and W.B. Jacoby, 1974. Glutathione Stransferases. The fiist enzymatic step in mercapturic acid formation. J. Biol. Chem., 249, 7130.
I
P.mo.437
ease
ability in the conk s
oil in v
a
Buben, I., Karmazin, 16, T~oj&nkov$ J. and Novl, D. State Institute for Drug Control, &otitova
48, 100 41 Prague IO, Czechoslovakia
The preparations of Thuja officinalis were used in the form of extracts or tinctures in the treatment of rheumatism and gout, in dermatology for removing of various kinds of warts and condylomas. When applied orally as abortives or anthehnintics, a potent toxic effect can be manifested by serious gastroenteritis, tonic and clonic convulsions, unconsiousness, heavy damage of kidneys and liver. Wooden dust, especially that of Thuja plicata, releases histamin from pulmonary tissue producing thus asthmatic attacks. An antibiotic activity of thujaplicin is even mentioned in the literature. Recently, an increasing attention has been paid to Thuja occidentalis due to its antiviral and imunostimulatory properties. In our experiments species of Thuja occidentalis L., T. occidentalis L., cv. Malonyiana, T. orientalis L., T. plicata
were observed. In ali of them, the content and composition of essential oil in leaves and in wood nomically different localities has been examined during one year. The content of essential oils in T. s sp. on individual localities was different with maximum values /5,25% W/v/ from June to December. in the wood was signilicantly lower /max. @SOW/. From other species the highest content achieved in T. Standishi /3,65-5,0%/, T. plicata /2,5-4,72%/, T. occidentalis, cv. / and T. orientalis /0,53-l,O%/. Throughout the examined period a lower content of been equally found in the above mentioned species. ter on we have tried to identify at least major components of essential oils by means of gas chromatography. T. orientalis sp. from 2 localities and T. plicata from 1 locality, /I-thujon in the concentration of 100-200 of the drug was proved to be the main component of essential oils. Other components in the number up to 50, mostly a- and Bpinene, camphene, thujaplicine and thujylalcohol are previling especially in Thuja Standishii.
Low, K., S.Y., Gwee, M.C.E. and Yuen *, R. of Pharmacoiog~ and * Biochmnistty, Facu& of Medicine, National University of Singapore, Lower Kent Ridge Road Singapore 0511, Singapore
The crude venom of Synanceja horrida has been reported to cause death within thirty seconds in rabbits treated with a dose of 300 pg/kg intravenously. Preliminary studies to determine the neuromuscular effects of the venom and its partially purified fK4CtiOAS WCR carried Out. Venom extrac& from ti;i Jorsal spines of S. horrida was passed through a Sephecryl column. The fraction exhibiting lethal activity in mice was further purified by passing it through a Diethylamino Ethyl Sepharose (DEAE) column. The lethal fraction thus obtained, DEAE lethal fraction (DLF) was then tested for effects on neuromuscular transmission and muscle contractility on indirectly stimulated mouse hem&diaphragm preparations. Activity showed better correlation with protein content of the fraction than with it’s dry weight, hence protein concentration was used as an index throughout the experiments. The mouse hem&diaphragm was mounted in Krebs solution gassed with 5% CO,/958 0s. Supramaximal nerve evoked twitches were elicited via stimulation of the motor nerve with square wave pulses of 0.2 msec duration at a frequency of 0.1 Hz. DLF caused an immediate contracture accompanied by a transient increase in the height of twitches. This was followed by a progressive blockade of the twitches as well as a gradual decrease in the musle tone. The extent and rate of neuromuscular blockade caused by DLF was concentration dependent (see table). In all the experiments, no apparent recovery of the preparation was observed up to 4 hours after the DLF was washed out. Cont. of DLF
Time to 20% block (mi@
Time to 50% block @inI
Time to 90% block (mw
Number of Experiments
7.3* 3 21.8* 6 133.0 f 10
13.4* 5 42.6+ 8 263.2 + 20
29.3* 8 88.2 f 27 >360
6 5 5
The results indicate that the venom of S. horrida has an irreversible neuromuscular blocking action on the diaphragm and may produce it’s rap’X! Iatai action by causing resi;;ratory pamlysis in timals. Further experiments remain to be doAe using the purified protein (after h&h performance liquid chromatography), as well as experiments with the venom preparations in whole animals.
itisnof rat liver
cytosofk
ione
Merlos, M., Sgnchez, R.M., Camarasa, J. and Adze& T. Lubotatqv of Pharmacology and Pharmacognosy. School oj Pharmacy, University of Barcelona, Zona Univetsitatia de Pedralbes s/n, 08028 Barcelona, Spain
Glutathione S-transferases (GST) are a family of cytosolic multifunctional enzymes with an important role in detoxification processes. They catalyse the conjugation of electrophilic xenobiotics with the sulfhydryl moiety of glutathione (GSH) (Kaplowitz, 1980). In this study the “in vitro” inhibitory potencies of different plant flavonoids on rat liver cytosolic GST activity were compared. The GST activity was assayed according to Habig et al. (1974) by using 1-chloro-2&dinitrobenzene (CDNB) as substrate. The IC,, values varied over a 30-fold range. In a general manner, the order of potency found was flavonols > flavones > flavanonols. Flavone nucleus alone provided only mild inhibition (IC, = 0.93 mM). Activity was shown to depend on 1) the pattern of hydroxylation: Thus, the presence of both hydroxyl groups in 3’,4’ provided enhanced activity in flavonols and flavones (IC, = 31 and 42 PM for quercetin and luteolin, respectively). 4’-hydroxylated analogous were less potent (IC, = 76 and > 200 FM for kaempferol and apigenin). 7-substitution by a sugar moiety decreases activity (7-glucosyl-luteolin, IC, = 210 @I). Additional 2’-hydroxylation of morin increased activity slightly (I& = 41 PM) in comparison with kaempferol. 2) the presence of a double bond in C-2,C-3: A 20-fold loss of activity was obtained with the flavanonol taxifolin compared with its analogous flavonol quercetin. Therefore, the planarity of benzopyrone ring seems to be an important requirement for inhibition. The kinetics of inhibition of quercetin, the strongest inhibitor found, revealed a non competitive profile versus both GSH and CDNB, with apparent Hi values of 75 and 60 FM, respectively. Querc&n was shown to inhibit GSH conjugation with several other electrophilic substrates, such as 1,2-dichloro4-nitrobenzene and p-nitrobenzyl chloride. On the contrary, an activation of GSH - 1,2-epoxy-3-(p-nitrophenoxy)propane conjugation was noted. A selective interaction with one or several of the isoenzymes which possess GST activity is proposed. eferences N. Kaplowitz, 1980, Physiological significance of glutathione S-transferases. J. Physioi, 239, G439. Habig, W.H.. Pabst, M.J. and W.B. Jacoby, 1974. Glutathione Stransferases. The fiist enzymatic step in mercapturic acid formation. J. Biol. Chem., 249, 7130.
I
P.mo.437
ease
ability in the conk s
oil in v
a
Buben, I., Karmazin, 16, T~oj&nkov$ J. and Novl, D. State Institute for Drug Control, &otitova
48, 100 41 Prague IO, Czechoslovakia
The preparations of Thuja officinalis were used in the form of extracts or tinctures in the treatment of rheumatism and gout, in dermatology for removing of various kinds of warts and condylomas. When applied orally as abortives or anthehnintics, a potent toxic effect can be manifested by serious gastroenteritis, tonic and clonic convulsions, unconsiousness, heavy damage of kidneys and liver. Wooden dust, especially that of Thuja plicata, releases histamin from pulmonary tissue producing thus asthmatic attacks. An antibiotic activity of thujaplicin is even mentioned in the literature. Recently, an increasing attention has been paid to Thuja occidentalis due to its antiviral and imunostimulatory properties. In our experiments species of Thuja occidentalis L., T. occidentalis L., cv. Malonyiana, T. orientalis L., T. plicata
were observed. In ali of them, the content and composition of essential oil in leaves and in wood nomically different localities has been examined during one year. The content of essential oils in T. s sp. on individual localities was different with maximum values /5,25% W/v/ from June to December. in the wood was signilicantly lower /max. @SOW/. From other species the highest content achieved in T. Standishi /3,65-5,0%/, T. plicata /2,5-4,72%/, T. occidentalis, cv. / and T. orientalis /0,53-l,O%/. Throughout the examined period a lower content of been equally found in the above mentioned species. ter on we have tried to identify at least major components of essential oils by means of gas chromatography. T. orientalis sp. from 2 localities and T. plicata from 1 locality, /I-thujon in the concentration of 100-200 of the drug was proved to be the main component of essential oils. Other components in the number up to 50, mostly a- and Bpinene, camphene, thujaplicine and thujylalcohol are previling especially in Thuja Standishii.
Low, K., S.Y., Gwee, M.C.E. and Yuen *, R. of Pharmacoiog~ and * Biochmnistty, Facu& of Medicine, National University of Singapore, Lower Kent Ridge Road Singapore 0511, Singapore
The crude venom of Synanceja horrida has been reported to cause death within thirty seconds in rabbits treated with a dose of 300 pg/kg intravenously. Preliminary studies to determine the neuromuscular effects of the venom and its partially purified fK4CtiOAS WCR carried Out. Venom extrac& from ti;i Jorsal spines of S. horrida was passed through a Sephecryl column. The fraction exhibiting lethal activity in mice was further purified by passing it through a Diethylamino Ethyl Sepharose (DEAE) column. The lethal fraction thus obtained, DEAE lethal fraction (DLF) was then tested for effects on neuromuscular transmission and muscle contractility on indirectly stimulated mouse hem&diaphragm preparations. Activity showed better correlation with protein content of the fraction than with it’s dry weight, hence protein concentration was used as an index throughout the experiments. The mouse hem&diaphragm was mounted in Krebs solution gassed with 5% CO,/958 0s. Supramaximal nerve evoked twitches were elicited via stimulation of the motor nerve with square wave pulses of 0.2 msec duration at a frequency of 0.1 Hz. DLF caused an immediate contracture accompanied by a transient increase in the height of twitches. This was followed by a progressive blockade of the twitches as well as a gradual decrease in the musle tone. The extent and rate of neuromuscular blockade caused by DLF was concentration dependent (see table). In all the experiments, no apparent recovery of the preparation was observed up to 4 hours after the DLF was washed out. Cont. of DLF
Time to 20% block (mi@
Time to 50% block @inI
Time to 90% block (mw
Number of Experiments
7.3* 3 21.8* 6 133.0 f 10
13.4* 5 42.6+ 8 263.2 + 20
29.3* 8 88.2 f 27 >360
6 5 5
The results indicate that the venom of S. horrida has an irreversible neuromuscular blocking action on the diaphragm and may produce it’s rap’X! Iatai action by causing resi;;ratory pamlysis in timals. Further experiments remain to be doAe using the purified protein (after h&h performance liquid chromatography), as well as experiments with the venom preparations in whole animals.