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Pharmacodynamic study of Hypericum perforatum L.
Phytomedicine, Vol. 7(6), pp . 449-453
© Urban & Fischer Verlag 2000 htt p://www.urbanfischer.de/j ournals/phytomed
Pharmacodynamic study of Hypericum perforatum L. V. jakovljevic' , M . Popovic", N. Mimica-Dukic/, A. Sabol and Lj. Cvozdenovic' Institute of Pharm acology, Toxicology and Clinical Pharmacology, Faculty of Medicine, University of Novi Sad, Yugoslavia . Institute of Chemistry, Faculty of Sciences, University of Novi Sad, Nov i Sad, Yugoslavia. 3 Institute of Surgery, Faculty of Medicine, University of Novi Sad, Yugoslavia 1
2
Summary The effects of Hypericum perforatum 1. (Hypericaceae) crude ethanol extract (A), ethyl acetate extract (B), aqueous extract (C) and infusion (I), on pentobarbital induced sleeping time, intestinal motility, and their analge sic activity, have been investigated. Extracts A and B exhibited significant stimulatory and antidepressant effects on the CNS. Both extracts prolonged sleep, increasing time up to more than 25 min. The antidepressive activity of extract A was also achieved by significant reduction of the myorelaxant activity of diazepam. Extract B exhibited stro ng analgesic activity reducing abdominal stretching induced by acetic acid by nearl y 50 %. Extracts A, Band C exhibited spasmolytic activity, significantl y reducing intestine motility. Key words: Flavonoids, forced-motor activity, hypericin, Hypericum perforatum L., pentobarb ital-induced sleep, spasmolytic activity.
Introduction Hypericum perforatum 1. (Hypericaceae) is a wellknown medicinal plant, used in folk-medicine to promote wound and burn healing. The plant is used against rheumatism, gou t, diarrhea and recently against mild viral infections and depre ssion (Bornbardelli and Morazzoni, 1995, Ma lamas and Marselos 1992, Serkedijeva et al. 1990). Altho ugh the antidepressant activity of H. perforatum extracts has been demonstrated and confirmed by recent clinical trials (Linde et. al., 1996 , Cod, 1996 ), neither the mechanism of antidepressant activity nor the con stituents respo nsible for th is effect are known (Holzl, 1993). Th e most cited mechan ism of action is the inhibition of mon oaminooxid ase (MAO) and catechol-O-methyltran sferase (COMT), enzymes respon sible for cata bolism of biological amines. Mo dulatory effects on inflamm ation mediat ors such as cytokines, or th e expression of the serotonin receptors und er stimulation, have also been considered (Bladt and Wagner 199 3, Muller and Rossol, 1993).
Although, at the beginn ing, hypericin and hypericinlike substances were cons idered the main antidepressant, recent studie s have shown th at a more polar fraction, consisting of flavon oids glycosides, tannins and procyanidins is also active (Demisch et al., 1989, Wagner and Bladt 1993) . Most pharm acological tests on H. perforatum are relat ed to the examin at ion of the behaviour of mice in an unknown environm ent (Winterhoff et al., 1995, 1993). The results obtai ned suggest a cent ral stimulating effect of the H. perforatum extracts. In order to provide more infor mat ion about th e pharmacological activity of H. perforatum, the effect of different H. perforatum extra cts on pentob arbital-indu ced sleep, forced-motor activity, intestina l motility, and their ana lgesic activity were investigated.
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Materials and methods Plant material
Hypericum perforatum L. (Hypericaceae) was collected from Vlasina Lake in Eastern Serbia during June 1995. For pharmacological study, the aerial part was used. The voucher specimen was authenticated by Dr. Boza Pal and deposited in the Botany Department, Faculty of Natural Sciences, University of Novi Sad.. Preparation of plant extract:
Infusion: 50 g of dry, powdered plant material were infused for 15 min. in 500 ml hot water. The leaves were separated by centrifuge and the supernatant was filtered. The aqueous extract was concentrated in vacuo, and used in experiment as infusion - L Extraction procedure: 200g of powdered plant material was extracted using 95 % ethanol in a Soxhlet apparatus for 3h. The solvent was removed under reduced pressure. The remaining water extract was fractioned into petroleum ether-, chloroform-, ethyl acetate-, nbutanol-, and water-solubles (Markham, 1989; Harborne, 1984; Popovic et aI., 1999). The qualitative composition of each extract was examined using TLC, along with reference substances (Maisenbacher and Kovar, 1992; Wicht!, 1995; Harborne, 1984). For further studies, dry residues of the crude ethanol (A), ethyl acetate (B) and water (C) extracts were used. The total hypericin content of each extract was determined spectrophotometrically. Dry extracts were resolved in ethanol and absorbance was measured at 592 nm (Southwell and Campbell, 1991). Biological Tests
These experiments were carried out on Swiss albino mice (both sexes, b. w. 30-50 g each). The animals were kept under constant environmental conditions (room temperature 21 ± 1°C, humidity 55 ± 1.05 % with light period of 14 hours and dark period of 12 hours). Animals were treated orally with 0.1 % (w/v) of the aqueous solutions of each extract for 12 days, successively. The control group received water. For intestinal motility and pentobarbital-induced sleep time, five groups of six mice were used. For testing forced motor activity and analgesic activity, ten mice per group were used. Thirty min. before testing, animals received 2 doses 1 % aqueous solution (10 ml/kg body wt.) of each extract. The control group received water. Pharmacodynamic study Pentobarbital-induced sleeping time
After thirteen days, animals received 1 % aqueous solutions i.p, (10mllkg body wt.) of each extract, the con-
trol group receiving only water, and thirty minutes later sodium pentobarbital was injected 40 mg/kg body wt. i.p. The time at which animals lost right reflex was registered as sleeping induction. The time between the loss and regaining of the righting reflex was measured as sleeping time (Gay, 1965). Intestinal motility
Thirty minutes after extracts or water administration mice received 10 % activated charcoal in 5 % agar suspension (10 mllkg body wt.) p.o. and were sacrificed 60 min later. The distance traveled by medical charcoal from blind gut was measured (Wagener et al., 1976). Forced motor activity
Thirty minutes after extracts (1 % i.p., 10 mllkg body wt.) or water administration animals received i.p. diazepam (2 mg/kg body wt.), The test for forced motor activity was conducted on mice trained to maintain equilibrium on a rotating rod revolving at 14 revolutions per minute. The animals that stayed for 5 minutes on the rotating rod were used for experiment. The selected animals were subjected to the rotating rod test at 10,20, 40, 50 and 60 minutes after diazepam was administrated i.p. (2.0 mg/kg body wt.). Each animal was subjected to three consecutive trials and mean time of maintaining equilibrium was recorded ( Ritschel et al., 1975). Analgesic activity
Analgesic activity was measured using of "the writhing test". Animals received 10 mg/kg body wt, 3 % solution of CH3COOH i.p. and the number of writhings was registered (Turner, 1965). Writhing is defined as a stretch, torsion to one side, drawing up of a hind leg, retraction of the abdomen, and opisthotonus, so that the belly of the mouse touches the floor. Narcotic analgesics, non-narcotic analgesics and antihistamines may inhibit writhing. Each value represents the mean ± SEM of 6-10 albino mice/group. Significance of differences with respect to the control group was evaluated using the Student ttest, p* ::::; 0.05.
Results Pentobarbital-induced sleeping time
Animals pretreated with extracts A and B had significantly prolonged sleep-inducing time, to more than 25 min. Extract C also prolonged both sleep-inducing time and duration of pentobarbital sleeping time, but the effect was not statistically significant (Table 1).
Pharmacodynamic study of Hypericum perforatum L.
451
Table 1. The effectof H. perjoratum extracts on pentobarbital-induced sleeping time and intestinal motility in mice GROUP (n=5)
SLEEP INDUCTION TIME (min)
SLEEPING TIME (min)
INTESTINAL MOTILITY (mm)
13.5 ± 6.5 13.0 ± 4.0 > 25" > 25':' 17.5 ± 7.0
45.0 ± 10.5 32.0 ± 9.5
48 ± 17 111 ± 64.5 165 ± 47.0" 127 ± 29.3" 141.3 ± 36.0*
CONTROL INFUSION-I EXTRACTS-A EXTRACTS-B EXTRACTS-C
50.5
±
14.0
*p:::; 0.05
Table 2. The effect of H. perforatum extracts on forced - motor activity in mice. The mean time of equilibrium maintenance is shown in seconds. TIME AFTER DIAZEPAM (2.5 mg/kg) ADMINISTRATION (min) GROUP CONTROL INFUSION -I EXTRACT-A EXTRACT-B EXTRACT-C
10
20
30
170.6± 94.5 197.1±128.3 70± 50.5 107.8±112.8 285± 26" 300* 142.5±107.8 150± 99.5 127.5± 57.6 257.5± 37.5
257.7± 58.3 183±122 300* 195± 88.7 275± 47
40 279.4± 58.3 184.5±121 300" 255± 49 277± 39.1
50
60
300 195±107 300* 300 285± 35
300 195±107 300* 300 300
*p:::; 0.05
Table3. The effectof H. perjoratum extracts on pain induced by 3 % CH3COOH. Time period after 3 % AcOH was injected (min) GROUP
5 - 20 ( I)
CONTROL INFUSION -I EXTRACT-A EXTRACT-B EXTRACT-C
17.8±5.4 10.8 ± 7.2 8.8 ± 2.9* 17.28±5.4 12.0 ± 4.3
50 % during the first time period. The total Reizing's number was significantly reduced during the second time period, although the reduction was not statistically significant.
20 - 35 ( II) 7.5 ± 4.2 4.25 ± 3.4 3.5 ± 2.5 7.5 ± 4.2 3.75 ± 2.4
*p:::; 0.05
Intestinal motility
All three extracts A, B, and C of Hypericum perforatum reduced intestinal motility in mice. The effect was most noticeable with extracts Band C. Forced-motor activity
The effect of H. perforatum extracts on forced - motor activity is shown in Table 3. Animals pretreated with extract A showed significantly reduced myorelaxant activity of diazepam. Analgesic activity
Infusion I and extract C tended to reduce the Reizing's number in mice, but a significant decrease was obtained only in animals pretreated with crude ethanol extract A, which reduced Reizing's numbers by nearly
Discussion All extracts examined exhibited strong antidepressant activity, by shortening sleep induction and duration of pentobarbital induced sleeping time. The obtained results are in accordance with several recent clinical trials, which compared the effects of pharmaceutical preparations of H. perjoratum with placebo and common antidepressants (Harsgen et. al. 1994; Harrer et. al. 1994; Martinez et. al. 1994; Woelk et. al. 1994 ). Antidepressant activity was also achieved with the crude EtOH extract (A) in the "rotating rod" test of forced-motor activity, in which extract A reduced significantly the myorelaxant activity of diazepam. Recently, Butterweck et, al. (1996), using the "forced swimming" test, examined different fractions of methanolic extracts of H. perforatum (LI 160) and found that fractions rich in flavonoid compounds as well as those rich in naphthodianthrones, such as pseudohypericin and hypericin, were remarkably active in prolonging the swimming period. In our experiment, both classes of the above constituents were identified in crude ethanol extract A. In extract B, hypericin-like compounds and some flavonoids were dominant, whereas in extract C, polar flavonoid glycosides remained. The highest hypericin content was found in ex-
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V. ]akovljevic et al.
tract B (8.34 mg/g) and the lowest in infusion I (1.23 mg/g) and extract e (0.98 mg/g). In crude ethanol extract A, hypericin content was 3.21 mg/g. Significantly reducing the intestinal motility, extracts Band e exhibited spasmolytic activity. The spasmolytic activity of certain flavonoids has been reported previously (Van der Broncke et. al., 1982). In our previous study, we found that extracts of Mentha longifolia L., rich in phenolic acids, possess strong spasmodic activity, whereas the flavonoid fraction showed spasmolytic activity (Mimica-Dukic et. al., 1996). In the current experiment, flavonoid aglycones and glycosides are presumed to be responsible for the spasmolytic action of H. perforatum. It was reported previously that the flavonoids chrysin, morin and rutin, showed significant analgesic activity when tested in mice using the acetic acid writhing test (Pathak et. al., 1991). In the present experiment, the crude ethanol extract (A) reduced significantly the number of abdominal stretches induced by acetic acid. Infusion I and extract e also reduced the number of stretches, but the effect was not statistically significant. Only in the group pretreated with extract B, in which the majority of hypericin-like compounds were present, did the number of abdominal stretches remain at the control level. Because analgesic activity is attributed to the central depressant activity, the results obtained suggest that hypericin-like compounds are probably responsible for the antidepressant effect of H.
perforatum. In conclusion, the results of the present pharmacodynamic study confirmed recently published data on the antidepressant effects of H. perforatum. In our experiment, the highest antidepressant and stimulative effects on eNS were achieved with the ethanol (A) and ethyl acetate (B) extracts. The last one also exhibited significant analgesic activity. In addition, extracts A, Band e showed spasmolytic activity on the smooth muscle of the gastrointestinal tract. The results obtained suggest that hypericin and hypericin-like constituents could be responsible for antidepressant and analgesic activity, while the spasmolytic activity of H. perforatum probably depends on flavonoid content. Acknoledgements This work was carried out with the financial support of the Serbian Ministry of Sciences and Education.
References Bladt S. and Wagner H.: MAO inhibition by fractions and constituents of hypericum extract, Nervenheilkunde, 12/6:
349-352, 1993. Bombardelli E. and Morazzoni P.: Hypericum perforatum 1. Recent Study, Fitoterapia 66: 43-68, 1995. Butterweck Winterhoff H., Schulz V., and Nahrstedt A.: Pharmacological in vivo testing of fractions obtained from Hypericum perforatum 1. 44th Annual Congress of the Society for Medicinal Plant Reserch, Prague, sept. 3-7, 65,
v.,
1996. Cod J.: Natural Product Formulations Available in Europe for Psychotropic Indications, Psychopharmacology bulletin, 31 (4): 745-751, 1995. Demisch L., Demisch K. and Gerbaldo H.: Fluvoxamine Melatonin Stimulation Test (Fmst) in Patients with Depressive Disorders, Pharmacopsychiatry, 21(6): 420-421,
1988. Gay W.J.: Methods of Animal Experimentation, 465-466, Academic Press, New York, London, 1965. Hansgen K.D., Vesper J., Ploch M.: Multicenter doubleblind study examing the antidepressant effectiveness of the H. perforatum extract LI 160, J. Geriatr. Psychiatry Neurol. 7
(1): 12-14, 1994. Harborne J. B.: Phytochemical methods. Sec edition. 89-100. Chapman and Hall, London, New York, 1984. Harrer G., Hubner W. D., Podzuweit H.: Effectiveness and tolerance of the hypericum extract LI 160 in comparison with imipramine : Randomized double-blind study with 135 outpatients. J. Geriatr. Psychiatry Neurol. 7 (1): S, 24-
28,1994. Holzl J.: Inhaltsstoffe und Wirkmechanismen des johanniskrauts. Zeitschrift fur Phytotherapie, 14: 255-64, 1993. Linde K., Ramirez G., Mulrow CD., Pauls, A., Weidenhammer W. and Melchart, D.: St. John's wort for DepressionAn Overview and Metaanalysis of Randomized Clinical Trials, British Medical Journal 313 (7052): 253-258,
1996. Maisenbacher P and Kovar K.A: Analysis and stability of Hyperici oleum. Planta Med. 58: 351-354, 1992. Malamas M., Marselos M.: The Tradition of Medicinal Plants in Zagori, Epirus (Northwestern Greece), Journal of Ethnopharmacology 37(3): 197-203, 1992. Markham K.R. In: Methods in Plant Biochemistry edited by Harborne J. B. and Dey P. M. 193-237, Academic - Press, London, 1989. Martinez B., Kasper S., Ruhrmann S. and Moller H.J.: Hypericum in the treatment of seasonal affective disorders. J. Geriatr. Psychiatry Neural. 7 (1): 29-33, 1994. Mimica-Dukic N. Biochemical Investigations of Secondary Biomolecules in Mentha species, pp. 46-49, PhD Thesis, Faculty of Sciences, University of Novi Sad, Novi Sad, 1993. Mirnica-Dukic N., jakovljevic V., Popovic M., Casic O. and Szabo A.: Pharmacological Study of Mentha Longofolia phenolic extracts, International Journal of Pharmacognosy
34:359-364,1996. Muller W.E.G., Rossol R.: Influence of hypericum extract on the expression of serotonin receptors, Nervenheilkunde
12(6): 357-358, 1993.
Pharmacodynamic study of Hypericum perjoratum L. Pathak D., Pathak K. and Singla A.K.: Flavonoids as medicinal agents-Recent advances, Fitoterapia 62: 371-389, 1991. Popovic M., Kaurinovic B., Mimica-Dukic N., VojinovicMiloradov M. and Cupic V.: Combined effect of plant extracts and xenobiotics on liposomal lipid peroxidation. Part 1. Marigold extract-ciprofloxacin-pyralene. Oxidation Communications 22 (4): 487-494, 1999. Ritschel A.W., Ring E.P., and Siegel E.G.: Biopharmaceutical Factors Influencing LDso , Arzneim. Forsch (Drug Res) 25(6): 853-856, 1975. Serkedjieva J., Manolova N., Zgorniak-Nowosieleska 1. and Zawilinska B.: Antivirial activity of the infusion (SHS-174) from flowers of Sombunus Nigra L., aerial parts of Hypericum perforatum L., and roots of Saponaria officinalis, against influenza and herpes simplex viruses. Phytotherapy Research 4 (3): 97-100, 1990. Staffeldt B., Kerb R., Brockmoller ]., Ploch M. and Roots 1.: Pharmacokinetics of hypericin and pseudo hypericin after oral intake of the Hypericum perforatum extract LI 160 in healthy volunteers, Nervenheilkunde 12(6): 331-338, 1993. Southwell LA., and Campbell M.H.: Hypericin content variation in Hypericum perforatum in Australia. Phytochemistry 30 (2): 475-478,1991. Turner R.A.: Screening Methods in Pharmacology, Academic Press, New York, 89,298, 1965.
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Van der Broncke C, Lemli]. and Lamy ].: Action spasmolitique des flavones de differentes especies de Thymus, Plant. Med. Phytother. 16: 310-317,1982. Wagner H. and Bladt S.: Pharmaceutical quality of hypericum extracts, Nervenheilkunde, 12(6): 362-366, 1993. Wagener H.H., Fountine R. and Neuman B.: Pharmakologie "Essentieller" Phospholipide (EPL), Arzneim-Forsch. (Drug Res.) 26: 1733, 1976. Wichtl, M.: Herbal Drugs and Phytopharmaceuticals. 273-275. Medpharm, Scientific Publishers, Stuttgart and CRC Press, Boca Raton, Ann Arbor, London, Tokyo, 1994. Winterhoff H., Hambrugge M. and Vahlensieck U.: Pharmacological screening of Hypericum perforatum L. in animals, Nervnheilkunde 12(6): 341-345,1993. Woelk H., Burkard G. and Grunwald ].: Benefits and risks of the hypericum extract LI 160: Drug monitoring study with 3250 patients.]. Geriatr. Psychiatry Neural. 7(1): 34-38, 1994.
Address M. Popovic, Institute of Chemistry, Faculty of Science, University of Novi Sad, Trg Dositeja Obradovica 3, 21000 Novi Sad, Yugoslavia Fax No 381 - 21 - 55662 E-mail: [email protected]
© Urban & Fischer Verlag 2000 htt p://www.urbanfischer.de/j ournals/phytomed
Pharmacodynamic study of Hypericum perforatum L. V. jakovljevic' , M . Popovic", N. Mimica-Dukic/, A. Sabol and Lj. Cvozdenovic' Institute of Pharm acology, Toxicology and Clinical Pharmacology, Faculty of Medicine, University of Novi Sad, Yugoslavia . Institute of Chemistry, Faculty of Sciences, University of Novi Sad, Nov i Sad, Yugoslavia. 3 Institute of Surgery, Faculty of Medicine, University of Novi Sad, Yugoslavia 1
2
Summary The effects of Hypericum perforatum 1. (Hypericaceae) crude ethanol extract (A), ethyl acetate extract (B), aqueous extract (C) and infusion (I), on pentobarbital induced sleeping time, intestinal motility, and their analge sic activity, have been investigated. Extracts A and B exhibited significant stimulatory and antidepressant effects on the CNS. Both extracts prolonged sleep, increasing time up to more than 25 min. The antidepressive activity of extract A was also achieved by significant reduction of the myorelaxant activity of diazepam. Extract B exhibited stro ng analgesic activity reducing abdominal stretching induced by acetic acid by nearl y 50 %. Extracts A, Band C exhibited spasmolytic activity, significantl y reducing intestine motility. Key words: Flavonoids, forced-motor activity, hypericin, Hypericum perforatum L., pentobarb ital-induced sleep, spasmolytic activity.
Introduction Hypericum perforatum 1. (Hypericaceae) is a wellknown medicinal plant, used in folk-medicine to promote wound and burn healing. The plant is used against rheumatism, gou t, diarrhea and recently against mild viral infections and depre ssion (Bornbardelli and Morazzoni, 1995, Ma lamas and Marselos 1992, Serkedijeva et al. 1990). Altho ugh the antidepressant activity of H. perforatum extracts has been demonstrated and confirmed by recent clinical trials (Linde et. al., 1996 , Cod, 1996 ), neither the mechanism of antidepressant activity nor the con stituents respo nsible for th is effect are known (Holzl, 1993). Th e most cited mechan ism of action is the inhibition of mon oaminooxid ase (MAO) and catechol-O-methyltran sferase (COMT), enzymes respon sible for cata bolism of biological amines. Mo dulatory effects on inflamm ation mediat ors such as cytokines, or th e expression of the serotonin receptors und er stimulation, have also been considered (Bladt and Wagner 199 3, Muller and Rossol, 1993).
Although, at the beginn ing, hypericin and hypericinlike substances were cons idered the main antidepressant, recent studie s have shown th at a more polar fraction, consisting of flavon oids glycosides, tannins and procyanidins is also active (Demisch et al., 1989, Wagner and Bladt 1993) . Most pharm acological tests on H. perforatum are relat ed to the examin at ion of the behaviour of mice in an unknown environm ent (Winterhoff et al., 1995, 1993). The results obtai ned suggest a cent ral stimulating effect of the H. perforatum extracts. In order to provide more infor mat ion about th e pharmacological activity of H. perforatum, the effect of different H. perforatum extra cts on pentob arbital-indu ced sleep, forced-motor activity, intestina l motility, and their ana lgesic activity were investigated.
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Materials and methods Plant material
Hypericum perforatum L. (Hypericaceae) was collected from Vlasina Lake in Eastern Serbia during June 1995. For pharmacological study, the aerial part was used. The voucher specimen was authenticated by Dr. Boza Pal and deposited in the Botany Department, Faculty of Natural Sciences, University of Novi Sad.. Preparation of plant extract:
Infusion: 50 g of dry, powdered plant material were infused for 15 min. in 500 ml hot water. The leaves were separated by centrifuge and the supernatant was filtered. The aqueous extract was concentrated in vacuo, and used in experiment as infusion - L Extraction procedure: 200g of powdered plant material was extracted using 95 % ethanol in a Soxhlet apparatus for 3h. The solvent was removed under reduced pressure. The remaining water extract was fractioned into petroleum ether-, chloroform-, ethyl acetate-, nbutanol-, and water-solubles (Markham, 1989; Harborne, 1984; Popovic et aI., 1999). The qualitative composition of each extract was examined using TLC, along with reference substances (Maisenbacher and Kovar, 1992; Wicht!, 1995; Harborne, 1984). For further studies, dry residues of the crude ethanol (A), ethyl acetate (B) and water (C) extracts were used. The total hypericin content of each extract was determined spectrophotometrically. Dry extracts were resolved in ethanol and absorbance was measured at 592 nm (Southwell and Campbell, 1991). Biological Tests
These experiments were carried out on Swiss albino mice (both sexes, b. w. 30-50 g each). The animals were kept under constant environmental conditions (room temperature 21 ± 1°C, humidity 55 ± 1.05 % with light period of 14 hours and dark period of 12 hours). Animals were treated orally with 0.1 % (w/v) of the aqueous solutions of each extract for 12 days, successively. The control group received water. For intestinal motility and pentobarbital-induced sleep time, five groups of six mice were used. For testing forced motor activity and analgesic activity, ten mice per group were used. Thirty min. before testing, animals received 2 doses 1 % aqueous solution (10 ml/kg body wt.) of each extract. The control group received water. Pharmacodynamic study Pentobarbital-induced sleeping time
After thirteen days, animals received 1 % aqueous solutions i.p, (10mllkg body wt.) of each extract, the con-
trol group receiving only water, and thirty minutes later sodium pentobarbital was injected 40 mg/kg body wt. i.p. The time at which animals lost right reflex was registered as sleeping induction. The time between the loss and regaining of the righting reflex was measured as sleeping time (Gay, 1965). Intestinal motility
Thirty minutes after extracts or water administration mice received 10 % activated charcoal in 5 % agar suspension (10 mllkg body wt.) p.o. and were sacrificed 60 min later. The distance traveled by medical charcoal from blind gut was measured (Wagener et al., 1976). Forced motor activity
Thirty minutes after extracts (1 % i.p., 10 mllkg body wt.) or water administration animals received i.p. diazepam (2 mg/kg body wt.), The test for forced motor activity was conducted on mice trained to maintain equilibrium on a rotating rod revolving at 14 revolutions per minute. The animals that stayed for 5 minutes on the rotating rod were used for experiment. The selected animals were subjected to the rotating rod test at 10,20, 40, 50 and 60 minutes after diazepam was administrated i.p. (2.0 mg/kg body wt.). Each animal was subjected to three consecutive trials and mean time of maintaining equilibrium was recorded ( Ritschel et al., 1975). Analgesic activity
Analgesic activity was measured using of "the writhing test". Animals received 10 mg/kg body wt, 3 % solution of CH3COOH i.p. and the number of writhings was registered (Turner, 1965). Writhing is defined as a stretch, torsion to one side, drawing up of a hind leg, retraction of the abdomen, and opisthotonus, so that the belly of the mouse touches the floor. Narcotic analgesics, non-narcotic analgesics and antihistamines may inhibit writhing. Each value represents the mean ± SEM of 6-10 albino mice/group. Significance of differences with respect to the control group was evaluated using the Student ttest, p* ::::; 0.05.
Results Pentobarbital-induced sleeping time
Animals pretreated with extracts A and B had significantly prolonged sleep-inducing time, to more than 25 min. Extract C also prolonged both sleep-inducing time and duration of pentobarbital sleeping time, but the effect was not statistically significant (Table 1).
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451
Table 1. The effectof H. perjoratum extracts on pentobarbital-induced sleeping time and intestinal motility in mice GROUP (n=5)
SLEEP INDUCTION TIME (min)
SLEEPING TIME (min)
INTESTINAL MOTILITY (mm)
13.5 ± 6.5 13.0 ± 4.0 > 25" > 25':' 17.5 ± 7.0
45.0 ± 10.5 32.0 ± 9.5
48 ± 17 111 ± 64.5 165 ± 47.0" 127 ± 29.3" 141.3 ± 36.0*
CONTROL INFUSION-I EXTRACTS-A EXTRACTS-B EXTRACTS-C
50.5
±
14.0
*p:::; 0.05
Table 2. The effect of H. perforatum extracts on forced - motor activity in mice. The mean time of equilibrium maintenance is shown in seconds. TIME AFTER DIAZEPAM (2.5 mg/kg) ADMINISTRATION (min) GROUP CONTROL INFUSION -I EXTRACT-A EXTRACT-B EXTRACT-C
10
20
30
170.6± 94.5 197.1±128.3 70± 50.5 107.8±112.8 285± 26" 300* 142.5±107.8 150± 99.5 127.5± 57.6 257.5± 37.5
257.7± 58.3 183±122 300* 195± 88.7 275± 47
40 279.4± 58.3 184.5±121 300" 255± 49 277± 39.1
50
60
300 195±107 300* 300 285± 35
300 195±107 300* 300 300
*p:::; 0.05
Table3. The effectof H. perjoratum extracts on pain induced by 3 % CH3COOH. Time period after 3 % AcOH was injected (min) GROUP
5 - 20 ( I)
CONTROL INFUSION -I EXTRACT-A EXTRACT-B EXTRACT-C
17.8±5.4 10.8 ± 7.2 8.8 ± 2.9* 17.28±5.4 12.0 ± 4.3
50 % during the first time period. The total Reizing's number was significantly reduced during the second time period, although the reduction was not statistically significant.
20 - 35 ( II) 7.5 ± 4.2 4.25 ± 3.4 3.5 ± 2.5 7.5 ± 4.2 3.75 ± 2.4
*p:::; 0.05
Intestinal motility
All three extracts A, B, and C of Hypericum perforatum reduced intestinal motility in mice. The effect was most noticeable with extracts Band C. Forced-motor activity
The effect of H. perforatum extracts on forced - motor activity is shown in Table 3. Animals pretreated with extract A showed significantly reduced myorelaxant activity of diazepam. Analgesic activity
Infusion I and extract C tended to reduce the Reizing's number in mice, but a significant decrease was obtained only in animals pretreated with crude ethanol extract A, which reduced Reizing's numbers by nearly
Discussion All extracts examined exhibited strong antidepressant activity, by shortening sleep induction and duration of pentobarbital induced sleeping time. The obtained results are in accordance with several recent clinical trials, which compared the effects of pharmaceutical preparations of H. perjoratum with placebo and common antidepressants (Harsgen et. al. 1994; Harrer et. al. 1994; Martinez et. al. 1994; Woelk et. al. 1994 ). Antidepressant activity was also achieved with the crude EtOH extract (A) in the "rotating rod" test of forced-motor activity, in which extract A reduced significantly the myorelaxant activity of diazepam. Recently, Butterweck et, al. (1996), using the "forced swimming" test, examined different fractions of methanolic extracts of H. perforatum (LI 160) and found that fractions rich in flavonoid compounds as well as those rich in naphthodianthrones, such as pseudohypericin and hypericin, were remarkably active in prolonging the swimming period. In our experiment, both classes of the above constituents were identified in crude ethanol extract A. In extract B, hypericin-like compounds and some flavonoids were dominant, whereas in extract C, polar flavonoid glycosides remained. The highest hypericin content was found in ex-
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tract B (8.34 mg/g) and the lowest in infusion I (1.23 mg/g) and extract e (0.98 mg/g). In crude ethanol extract A, hypericin content was 3.21 mg/g. Significantly reducing the intestinal motility, extracts Band e exhibited spasmolytic activity. The spasmolytic activity of certain flavonoids has been reported previously (Van der Broncke et. al., 1982). In our previous study, we found that extracts of Mentha longifolia L., rich in phenolic acids, possess strong spasmodic activity, whereas the flavonoid fraction showed spasmolytic activity (Mimica-Dukic et. al., 1996). In the current experiment, flavonoid aglycones and glycosides are presumed to be responsible for the spasmolytic action of H. perforatum. It was reported previously that the flavonoids chrysin, morin and rutin, showed significant analgesic activity when tested in mice using the acetic acid writhing test (Pathak et. al., 1991). In the present experiment, the crude ethanol extract (A) reduced significantly the number of abdominal stretches induced by acetic acid. Infusion I and extract e also reduced the number of stretches, but the effect was not statistically significant. Only in the group pretreated with extract B, in which the majority of hypericin-like compounds were present, did the number of abdominal stretches remain at the control level. Because analgesic activity is attributed to the central depressant activity, the results obtained suggest that hypericin-like compounds are probably responsible for the antidepressant effect of H.
perforatum. In conclusion, the results of the present pharmacodynamic study confirmed recently published data on the antidepressant effects of H. perforatum. In our experiment, the highest antidepressant and stimulative effects on eNS were achieved with the ethanol (A) and ethyl acetate (B) extracts. The last one also exhibited significant analgesic activity. In addition, extracts A, Band e showed spasmolytic activity on the smooth muscle of the gastrointestinal tract. The results obtained suggest that hypericin and hypericin-like constituents could be responsible for antidepressant and analgesic activity, while the spasmolytic activity of H. perforatum probably depends on flavonoid content. Acknoledgements This work was carried out with the financial support of the Serbian Ministry of Sciences and Education.
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Address M. Popovic, Institute of Chemistry, Faculty of Science, University of Novi Sad, Trg Dositeja Obradovica 3, 21000 Novi Sad, Yugoslavia Fax No 381 - 21 - 55662 E-mail: [email protected]