however, desirable to produce medicines with standardized pharmaceutical quality. Further research on hyperforin, hypericins and flavonol glycosides, active.
Antidepressant Constituents of Hypericum perforatum Adolf Nahrstedt
Summary Several compounds from different structural groups and with different mechanisms of action see m to be responsible for the observed antidepressant efficacy of Hypericum extracts. Consequently, it is not useful to isolate single constituents from Hypericum crude drug material in order to use them therapeutically. Standardization on constituents that contribute to efficacy is, however, desirable to produce medicines with standardized pharmaceutical quality. Further research on hyperforin, hypericins and flavonol glycosides, active constituents of Hypericum herbal products, is needed.
Introduction Alcoholic extracts produced from the upper third of the flowering plant Hypericum perforaturn (family: Clusiaceae) belong to the most prescribed medicines in Germany. They are used for the treatment of mild and moderate depressions (Schwabe and Paffrat 1999). Recently new information emerged indicating that antidepressants, and among them Hypericum extracts, reduce pain in that they influence the chronic manifestation of pain (Anonymus 1(99). Only few serious side effects have been observed yet, one of which is the interference of Hypericum extracts with the pharmacokinetics of digoxin (lohne et al. 1(99), phenprocoumon and other synthetic drugs (Anonymus 1999). While aseries of compounds has been detected in the crude drug material (for recent reviews see: Nahrstedt and Butterweck 1997; Bombardelli and Morazzoni 1995), the active constituents are still a matter of debate. However, knowledge about the active constituents is aprerequisite of a rational herbal medicinal product in order to keep its quality at a high level. Briefly, pharmaceutical quality, which inc1udes standardization of the active constituents, is the basis of therapeutical quality of each medicinally used drug. Therefare increased efforts are being made to detect these compounds in extracts of Hypericum perforaturn and to establish their mechanisms of action. Phenylpropanes, flavonolglycosides, oligomeric flavanols, biflavones, xanthones, phlaroglucinols, an essential oil, some amino acids and naphthodianthrones have been isolated and were structurally elucidated from flowering Hypericum herb (Nahrstedt and Butterweck 1997). Out of these, the amino acids, the 144
phenylpropanes, oligomeric flavanols and the essential oil components do not seem to have antidepressant activity and, thus, are obviously not eonnected to the observed efficacy of the erude drug material. In the following, the present knowledge on the residual groups of compounds is briefly reviewed, with reference only to the most recent literature. Xanthones These compounds are typical eonstituents of the Clusiaceae; they exhibit strong
o
OH
1,3,6,7 -Tetrahydroxyxanthone Figure 39. Chemical structure of 1,3,6,7 - tetrahydroxyxanthone
MAO (monoamine oxidase) inhibiting properties (Suzuki et al. 1981, Schaufelberger and Hostettmann 1988). However, the concentration of xanthones in H. perforatum is low; kielcorin was found in the roots in an amount of ca. 0.01 %. 1,3,6,7-tetrahydroxyxanthone (Figure 39), found in trace amounts as the only xanthone of the upper parts of H. perforatum, exhibits a MAO-inhibiting activity of 95 u mol/L (Sparenberg et al. 1993). But in view of its very low concentration of approximately 0.0004% this xanthone derivative cannot be responsible for the antidepressive effieacy of the crude drug material. Biflavones 13,II8-Biapigenin and amentoflavone (13' ,II8-biapigenin) as shown in Figure 40 occur exclusively in the buds and blossoms of H. perforatum (Berghoefer and Hoelzl 1987; 1989). Amentoflavone competitively binds to the benzodiazepine receptors in vitro (Nielsen et al. 1988; Baureithel et al 1997); however, it was suggested that in vivo amentoflavone will not reach the central nervous system because it does not penetrate through the blood-brain barrier (Nielsen et al. 1988). On the other hand, using a monolayer of cultured brain capillary endothelial cells it was shown recently that energy-independent diffusion of amentoflavone through the monolayer is possible (Gutmann et al. 1999). Asedative effect was also observed with this compound in mice at an intraperitoneal dosage of 30-300 mg/kg (Brugisser et al. 1999). However, in patients treated with the usual oral dosage of 900 mg of Hypericum extract per day an amount of about 0.45 to 2.5 mg of amentoflavone will be administered, calculated on the content of 0.01 -
145
0.05% in the erude drug. These data are not in favor for amentoflavone as an aetive eonstituent. It is surprising that amentoflavone instead of I3,II8-biapigenin was used for these experiments, beeause the latter is ab out ten times more highly eoneentrated in the herb of H. perforaturn. Therefore, I3,II8-biapigenin eould be a more realistic eandidate for aetivity. However, in the foreed swimming test (see below) amentoflavone did not show aetivity (Butterweek 1997).
OH OH
I3',IlS Biapigenin (Amentoßavon)
HO
OH
HO
0
I3,llS Biapigenin
Figure 40. Chemical structures
OH
o OH 0
of 13,118 biapigenin and amentoflavone
Hyperforin The main representative of the phloroglucinol group of constituents is hyperforin (Figure 41). Together with adhyperforin it occurs exclusively in the generative parts of the plant, especially in the unripe fruits. Consequently, plant material collected at the end of the flowering period, when fruits are developing, will contain more hyperforin than material collected, as prescribed by the monographs, during full blooming. Hyperforin accounts for 2-5% in the erude drug material and can reach about 6% in some extracts. Although this eompound is quite unstable, especially in solution and when exposed to light and heat, it is present in many eommercial extraets but at largely varying concentrations of 0 6% (Melzer and Kolkmann 1998). Products of degradation recently detected are 2-methyl-3-buten-2-o1 (Figure 41) and two oxidation produets with an intaet hyperforin carbon skeleton (Trifunovie et al. 1998; Orth et al. 1999).
146
OH
R = H: Hyperforin
HO
R = CH3: Adhyperforin
:1 Methylbutenol
Figure 41. Chemical structure of hyperforin
Chatterjee and coworkers (1998) have recently pointed to hyperforin as the major non-nitrogenous metabolite of H. perforatum. They have shown that hyperforin in vitro inhibited serotonin-induced responses and uptake of neurotransmitters in peritoneal cells when hyperforin enriched CO2 extracts of the herb were tested. The pharmacological work was continued by Müller et a1. (1998), who detected in vitro inhibition of synaptosomal reuptake of serotonin, norepinephrine and dopamine and a down-regulation of cortical ß-adrenoceptors and 5-HT2-receptors after subchronic treatment of rats. These data made hyperforin a candidate responsible for effectiveness. Recently the compound was shown in vitro to inhibit serotonin uptake by elevating free intracellular sodium ions (Singer et al. 1999). However, hyperforin by far may not be the major antidepressant constituent nor the only one responsible for activity, as is sometimes suggested by advertisements and some meeting reports. It was shown by Chatterjee et a1. (1998) that the activity of pure hyperforin does not exp1ain the activity of a methanolic extract of the crude drug; the latter was more active in inhibition of synaptosomal uptake of various neurotransmitters than was pure hyperforin. This was especially pronounced for inhibiton of the reuptake of serotonin, dopamine and GABA. A clinical study which is often cited in favor of hyperforin being the active component was published recently (Laakmann et al. 1998). The study claimed to have demonstrated the relevance of hyperforin for clinical efficacy over a 42 day period. It showed a statistically significant difference between an extract with 5% hyperforin in reducing the Hamilton depression score towards placebo, and an extract with 0.5% hyperforin.
147
Unfortunately, the study suffers from several deficiencies: (i) The difference at day 0 in that study and the last day of treatment was 10.2 score points for the 5%, 8.5 points for the 0.5% extract and 7.9 points for placebo; thus the difference between both the high and the low hyperforin extracts in comparison to placebo was unusually small. (ii) The main objection is that no quantitative data are presented for additional constituents in the extracts such as flavonoids, hypericins, biflavones, which are all well measurable now by HPLC methods (eg. Butterweck et al. 1997); thus, it cannot be excluded that constituents other than hyperforin may have caused the smaIl differences in efficacy of both extracts. (iii) The important information is missing about how the two extraets were obtained. One may argue that both extracts were prepared from different batehes of emde drug material, in that for the 5% extract crude drug material with more unripe fruits was selected (see above). Consequently, the study did not "demonstrate a clear-cut relationship between hyperforin dose and antidepressant efficacy" as stated (Laakmann et al. 1998). The outcome is not eonvincing proof for the activity of hyperforin, but indieates that there are extraets whieh may show clinical effieaey, and others which may not. Hypericins The naphthodianthrones hypericin and pseudohypericin (Figure 42), as well as other derivatives, oecur in the flowers and leaves of the emde drug material at concentrations of 0.03 to 0.3%, depending on the developmental stage of the plant with significant variation (Cellarova et al. 1994). A fraction with hypericin and other compounds was recognized to inhibit MAO (Suzuki et al. 1984) but this effect eould not be eorroborated later with pure hypericin (Bladt and Wagner 1993). OB
0
OH
R = CH3 R
HO HO
OH
0
OH
Figure 42. Chemical structure of hypericin
148
= CH20H
Hypericin Pseudohypericil
Besides in vitro testing for antidepressant activity, several in vivo behavioural tests are available, one of which is the so-called Porsolt test or forced swimming test (porsolt et al. 1977), FST, which measures the activity of rats in escaping from a glass cylinder filled with water. The reduction of despair time (no movement of the rats) is used as a positive parameter. The FST is a weIl established animal model for the evaluation of antidepressant drugs, as it correlates weIl with their clinical efficacy (porsolt et al. 1978). The FST is used in a short term testing for a 24 hour period as weIl as in "chronic" testing for an 11-14 day period. This test was used for a bioassay guided fractionation of a commercially used Hypericum extract (Butterweck et al. 1997). Whi1e the entire extract was clear1y active with a so-called inverted U-shaped activity, two main active fractions were obtained, one of which contained hypericin, pseudohypericin and procyanidins as constituents (Butterweck et al. 1997). However, in the 24 hour test pure hypericin and pseudohypericin were not active at doses comparable to the entire crude extract; and only the exceptionally high dose of 0.23 mg/kg of hypericin was significantly active, whereas pseudohypericin indicated some non-significant activity at about 0.5 mglkg (Butterweck et al. 1998a). When the fraction of procyanidins, which was itself not active in the FST, was recombined with the hypericins, hypericin was significantly active at 0.028 mg/kg and pseudohypericin at 0.166 mg/kg, with both compounds again having an inverted U'-shaped activity. Interestingly, the activity was totally blocked by the dopamine reuptake inhibitor sulpiride. In the same paper it was shown that the procyanidin fraction of H. perforatum, as weIl as the pure procyanidins B2 and Cl, increase the solubility of both hypericins, which may lead to their better bioavailability (Butterweck et al. 1998a). However, in long tenn experiments (14 days) hypericin was active in the FST without supplementation of procyanidin B2 (Butterweck et al.; in preparation), indicating that when hypericin is applied chronically the endogeneous level will reach a pharmacodynamic concentration without solubilizer. When hypericin plus procyanidin B2 were administered to rats in a two week treatment a significant reduction in the corticosterone level in the rat' s serum was observed, whereas hypericin alone did not influence this parameter. However, hypericin alone or given with procyanidin B2 had no effect on the dopamine concentration in rat brain samples, but the entire crude extract significantly increased dopamine, as did the synthetic reference compound imipramine (Butterweck et al. 1998b). The influence of Hypericum extract and hypericin on the release of corticosterone, probably via corticotropin releasing factor, is currently under investigation. All data obtained by recent studies show that the hypericins exert in vivo aseries of effects which are in good agreement with an antidepressant activity. There is
149
no doubt that they belong to those constituents which are important clinically observed efficacy of Hypericum extracts.
for the
Flavonoids Previous reports suggest that flavonoids, which occur at 2 - 4% in the crude drug material, may be part of the constituents responsible for efficacy of Hypericum extracts: a flavonoid containing fraction in vitro inhibited monoamine oxidase (Sparenberg et al. 1993) and catechol-0-methyl transferase (Thiede and Walper 1993). However, no clear cut results were presented. During the bioassay guided fractionation of Hypericum extract, as described by Butterweck et al. (1997), a fraction 11 characterized by its content of flavonoids exerted considerable activity in the FST (Butterweck et al. 1997). This fraction was further purified and some flavonol glycosides such as hyperoside, quercitrin, isoquercitrin and miquelianin (Figure 43), as weIl as the flavanonol glycoside astilbin (Figure 43), were isolated and tested for activity in the FST at doses comparable to their application with the crude drug material (Butterweck et al. 2000). Except for quercitrin and astilbin, all flavonoids (eg. hyperoside, isoquercitrin and miquelianin) were significantly active in the FST in a 24 hour and 12 day treatment regime. It was shown that the three compounds did not induce hypermotility in the Open Field Test, indicating that the effects of the flavonoids in the FST are specific. The aglycone quercetin (Figure 43) of the active flavonol glycosides did not show activity in the FST when tested at 0.4 mglkg analogous to hyperoside. The diglycoside rutin was already obtained in a strongly enriched fraction during the fractionation of the entire extract (Butterweck et al. 1997); this fraction showed no activity in the FST.
~OH HO
HO
OH
""""~OH
OR
0-
OH R=H R a-L-rhamnosyl R ß-D-glucosyl R ß-D-galactosyl R ß-rutinosyl R ß-D-glucuronyl
= = = = =
Quercetin Quercitrin Isoquercitrin Hyperoside Rutin Miquelianin
Astilbin
Figure 43. Chemical structure of various flavonoids
150
0
rhamnosyl
The data obtained (Butterweck et al. 2000) indicate some certain flavonol 3-0glycosides as active constituents of Hypericum extract. The activity seems to be bound to the sugar moiety of the aglycone quercetin, in that its glucoside, its galactoside and, interestingly, its glucuronide are the active compounds in the series of tested flavonoids. It may be that these particular glycosides are absorbed from the intestine whereas others are not. Further studies will be necessary to obtain more insight into the structure activity relationship and mode of action of the flavonol glycosides. Conclusion As for many herbal medicinal products Hypericum extracts are characterized by a complex situation when regarding their active constituents. Nevertheless, continuous interdisciplinary research between pharmacologists and phytochemists has shed new light on this crude drug material. As for many herbal medicinal products, several compounds from different structural groups and with different mechanisms of action seem to be responsible for the observed antidepressant efficacy. Consequently, it does not make sense to isolate them from Hypericum crude drug material in order to use them therapeutically as isolated constituents, but their standardized presence in Hypericum extracts should be guaranteed to present medicines with standardized pharmaceutical quality. Research on the active constituents of Hypericum herbal products is by far not finished. For hyperforin in vivo studies are necessary and further studies on the mechanism of action of the hypericins and the flavonol glycosides have to be performed. In view of some newly recognized side effects (see Introduction) additional studies on toxicology, especially on interaction with other drugs, should be carried out. Pharmacokinetic data for the active components should be elaborated and clinical data to establish the suitable daily dose for human treatment should be collected. Acknowledgement: Thanks are due to Dr. V. Butterweck and Prof. H. Winterhoff (Dept. of Pharmacology, University of Münster) for continuous excellent cooperation in the field of Hypericum research. References Anonymus: Mit Johanniskraut gegen den Schmerz. Aerzte Zeitung (München) July 8, 1999_ Anonymus: Vorsicht bei Johanniskraut - Wechsel wirkungen mit Digoxin! Dtsch Apoth Ztg 1999; 139; 4718. Baureithe1 KH, Bueter KB, Engesser A, Burkard W, Schaffner W: Inhibition of benzodiazepine binding in vitro by amentoflavone, a constituent of various species of Hypericum. Phann Acl Helv 72; 1997; 153:157. Berghoefer R, Hoelzl, J: Biflavonoids in Hypericum perforatum; Part 1. Isolation of 13,II8biapigenin. Planta Medica 53; 1987; 215:216. Berghoefer R, Hoelzl J: Isolation of 13',Il8-biapigenin (amentoflavone) from Hypericum perforatum. Planta Medica 55; 1989; 91.
151
Bladt S, Wagner H: MAO-Hemmung durch Fraktionen und Inhaltsstoffe von Hypericum-Extrakt. Nervenheilkunde 12; 1993; 349:352. Bombardelli E, Morazzoni P: Hypericum perforatum. Fitoterapia 66; 1995; 43:68. Brugisser R, Burkard W, Jenck F, Gutmann H, Drewe J, Schaffner W: Effect of Hypericum extract and amentoflavone in a behavioural test of free exploration in the mouse. 10. Annual Meeting of the Society of Phytotherapy, Münster, 1999. Book of Abstracts p. 76. Butterweck V: Beitrag zur Pharmakologie und Wirkstoff-Findung in Hypericum perforatum. PhD Thesis. 1997; Univ. of Muenster, Germany. Butterweck V, Wall A, Liefländer- Wulf U, Winterhoff H, Nahrstedt A: Effects of the total extract and fractions of Hypericum perforatum in animal assays for antidepressant activity. Pharmacopsychiatry 30; 1997 (Suppl.); 117: 124. Butterweck V, Petereit F, Winterhoff H, Nahrstedt A: Solubilized hypericin and pseudohypericin from Hypericum perforatum exert antidepressant activity in the forced swimming test. Planta Medica 64; 1998a; 291 :294. Butterweck V, Nahrstedt A, Winterhoff H: Effects of Hypericum perforatum extract and isolated constituents on neurotransmitter concentrations and endocrine parameters. 46. Annual Congress of the Soc. Med. Plant Res. Vienna 1998b. Book of Abstracts p. J29. Butterweck V, Jürgenliemk G, Nahrstedt A, Winterhoff H: Flavonoids from Hypericum perforatum show antidepressant activity in the Forced Swimrning Test. Planta Medica 66; 2000; 2:6. Cellarova E, Daxnerova Z, Kimakova K, Haluskova J: The variability of the hypericin content in the regenerants of Hypericum perforatum. Act Biotechol14; 1994; 267:274. Chatterjee SS, Nöldner M, Koch E, Erdelmeier C: Antidepressant activity of Hypericum perforatum and hyperforin: The neglected possibility. Pharmacopsychiatry 31, 1998 (Suppl.); 7:15. Chatterjee SS, Bhattacharya SK, Wonnemann M, Singer A, Müller WE: Hyperforin as a possible antidepressant component of Hypericum extracts. Life Seiences 63; 1998; 499-510. Gutmann H, Brugisser R, Drewe J, Schaffner W: Transport of amentoflavone, a constituent of Hypericum perforaturn across the blood brain barrier. 10. Annual Meeting of the Society of Phytotherapy, Münster, 1999. Book of Abstracts p. 80. Johne A, Brockmoeller J, Bauer S, Mauerer A, Langheinrich M, Roots I: Pharmacokinetic interaction of digoxin with a herbal extract from St. Johns wort (Hypericum perforatum). Clin. Pharmcol. Ther. 66; 1999; 338:345. Laakmann G, Schüle C, Baghai T, Kieser M: St, Johns wort in mild to moderate depression: The relevance of hyperforin for clinical efficacy. Pharmacopsychiatry 31, 1998 (Suppl.); 54:59. Melzer M, Kolkmann R: Hyperforin im Johanniskraut. Dtsch Apoth Ztg 138; 1998; 4754:4762. Müller WE, Singer A, Wonnemann M, Hafner U, Rolli M, Schäfer C: Hyperforin represents the neurotransmitter reuptake inhibiting constituent of Hypericum extract. Pharmacopsychiatry 31. 1998 (Suppl.); 16:21. Nahrstedt A, Butterweck V: Biologically active and other constituents of the herb of Hypericum perforatum L. Pharmacopsychiatry 30; 1997; 129: 134. Nielsen M, Frokjaer S, Braestrup C: High affinity of the naturally-occurring biflavonoid, amentoflavon, to brain benzodiazepine receptors in vitro. Biochem Pharmacol 37; 1988; 3285:3287. Orth HCJ, Hauer H, Erdelmeier CAJ, Schmidt PC: Orthoforin: the main degradation product of hyperforin from Hypericum perforatum. Pharmazie 54; 1999; 76:77. Porsolt RD, Le Pichon M, Jalfre M: Depression: A new animal model sensitive to antidepressant treatments. Nature 266; 1977; 730:732. Porsolt RD, Anton G, Blavet N, Jalfre, M: Behavioural despair in rat: A new model sensitive to antidepressant treatments. Eur J Pharmacol47; 1978; 379:391.
152
Schaufelberger D, Hostettmann K: Chemistry and pharmacology of Gentiana lactea. Planta Medica 54; 1988; 219:221. Schwabe U, Paffrat D (Ed.): Arzneiverordnungsreport 1999. Berlin,Springer, 2000, p. 488 ff. Singer A, Wonne mann M, Müller WE: Hyperforin, a major antidepressant constituent of St. John's wort, inhibits serotonin uptake by elevating free intercellular Na+. J Pharrnacol Exptl 1her 290; 1999; 1363-1368. Sparenberg B, Demisch L, Hoelzl J: Untersuchungen über antidepressive Wirkstoffe von Johanniskraut. Pharrn Ztg Wiss. 6/138; 1993; 50:54. Suzuki 0, Katsumata Y, Oya M, Chari V, Vennes B, Wagner H, Hostettmann K: Planta Medica 42; 1981; 17:21. Suzuki 0, Katsumata Y, Oya M, Bladt S, Wagner H: Inhibition of mono amine oxidase by hypericin. Planta Medica 50; 1984; 272:274. Thiede H-M, Walper A: MAO- und COMT-Hemmung durch Hypericum Extrakte und Hypericin. Nervenheilkunde 12; 1993; 346:348. Trifunovic S, Vajs V, Macura S, Juranie N, Djarmati Z, Jankov R, Milosavljevic S: Oxidation products of hyperforin from Hypericum perforatum. Phytochemistry 49; 1998; 1305: 1313.
I
I
1
153
