Chemistry and Biology of Hypericum perforatum (St. John's Wort

Chapter 20

Chemistry and Biology of Hypericum perforatum (St. John's Wort)

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Hans D. Reuter Siebengebirgsallee 24, D-50939 Köln, Germany

Hypericum perforatum, besides the hypericins (protohypericin, pseudohypericin, cyclopseudohypericin, hypericin), contains additional supposed biologically active compounds such as hyperoside, rutin, quercitin, and chlorogenic acid. As mechanisms for the antidepressant action of Hypericium, an increase in the number of neurotransmitters, an inhibition of type A monoamine oxidase (MAO), an inhibition of catechol-O-methyl-transferase (COMT), a modulation of cytokine expressivity, hormonal effects, and photodynamic effects have been discussed. It is still not possible to assign the antidepressant effects of Hypericum perforatum to specific constituents. Following the inhibition of M A O and COMT by the hypericins, it would seem unlikely that inhibition of these enzymes is the main active antidepressant principle of Hypericum. Some experiments suggest that the flavonoids are also involved in the action of the total extract. From recent investigations it can be concluded that the dopaminergic system is involved in the activity of Hypericum extract.

St. John's wort has been used for the treatment of psychiatric disorders for centuries since the times of Paracelsus (1493-1541). St. John's wort was then described as "arnica for the nerves." Today St. John's wort is one of the most important psychotropic drugs. It is widely used in Germany and central Europe for the treatment of psychovegetative disturbances, light depressive states, anxiety and/or nervous restlessness, according to the drug monograph of the German drug regulatory authorities (1). St. John's wort originates from the wild in Europe and western Asia. The drug consists of the dried flowering tops. Particularly noteworthy are the yellow to yellowbrown flowers which under certain circumstances are still present in the form of cymes and whose petals are covered with numerous dark dots or streaks. Fifty to sixty stamens of each flower are usually fused into three groups. The often shriveled and folded green to brownish-green smooth, ovate-elliptic leaves are up to 3.5 cm long, with clearly visible

©1998 American Chemical Society

In Phytomedicines of Europe; Lawson, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

287

288 translucent dots. These dots represent oil glands, filled with voatile oil. Because of the refractive index of the oil glands differing from that of the other cells, the leaves seem to be perforated if they are looked at against the light. From this phenomenon the designation Hypericum perforatum is derived.


Constituents of Hypericum Herb The constituents of Hypericum herb (Figure 1) are 0.05-0.3% hypericin and hypericinlike substances, notably pseudohypericin, isohypericin and the protohypericins. The nothern European broad-leaved variety perforatum tends to have less of the hypericins than the southern European variety angustifolium D C , the concentration correlating well with the leaves oil gland counts. The special structure of the hypericins consisting of a system of conjugated double bonds is responsible for their red color and of the rather severe phototoxic skin reactions which may occur upon exposure to ultraviolet light after applying Hypericum oil topically (2). Among the antibiotic substances there are about 3% hyperforin, a phloroglucinol derivative structurally related to the bitter substances of hops (2). Furthermore, there are flavonoids, especially hyperoside and rutin, and 2,3,6,7tetrahydroxy-xanthone respectively. Hypericum also contains biflavones, particularly 13,118-biapigenin (0.26%), amentoflavone and small amounts of procyanidins. The essential oil (0.05 to 0.3%) consists of n-alkanes, especially C ^ H ^ cc-pinene and other monoterpenes. Additionally, there are up to 10% condensed tannins (2). When making extracts it must be remembered that hypericin and the pseudohypericin are released in very different amounts. Thus, in a hot infusion, 22% of pseudohypericin is released within the first 10 minutes, whereas only 6% of the hypericin is released, hi the subsequent 60 minutes a further 23% of the pseudohypericin, compared to a further 7% of the hypericin, is released. Experiments with extraction at various temperatures show that maximum release of pseudohypericin is achieved at 60-80°C, above which the yield decreases. Basically, this also applies to hypericin, but both the amount released and the temperature sensitivity are lower. With increased methanol concentration, the amount of hypericin extracted increases. Pseudohypericin increases with 80% methanol and decreases with 100% methanol to 30% of the 80% methanol value (3). Controlled Clinical Trials Despite the long use of Hypericum preparations, systematic investigation for its effectiveness against depression has been neglected until approximately 10 years ago. In contrast to the development of synthetic drugs, studies into the clinical efficacy and tolerability of Hypericum have been performed predominantely while pharmacological investigations were conducted merely as supplementary measures. To date, there have been about 30 controlled trials with Hypericum extracts, involving over 1500 depressive patients with daily dosages of 900 mg for between 28 and 42 days. They have confirmed the antidepressant action of Hypericum extracts, especially that of LI 160 or Jarsin. The extract LI 160 is characterized phytochemically as follows: 100 mg extract containing 300 pg hypericins. Trials comparing the effects of this preparation and maprotilin showed



289

HO

O

OH

HO

O

OH

R = CHg: Hypericin R = CH2OH: Pseudohypericin OH

OH

1,3,6,7-Tetrahydroxy-xanthone .OH

Quercetin R = H Hyperoside R = Galactose Quercitrin R = Rhamnose Isoquercitrin R - Glucose Rutin R = Rhanmose-Glucose HO

OH OH

HO

OH

13, II8-Biapigenin OH OH

Amentoflavone

Dimeric Procyanidine Figure 1. Constituents of Hyperici herba.



290 similar depression-relieving and mood-lifting effects in both products, whereas maprotilin showed a more pronounced sedative action (4, 5). Jarsin, with its action spectrum of slightly stimulative and very slightly sedative components, is very similar to imipramin (6). Despite proof of the therapeutic efficacy of St. John's wort in patients with mild depressions the mechanism of the antidepressant effect is still unclear. The latest issue of the Comission E drug monograph of the German drug regulatory authorities dates from 1984 (1). This monograph recommends as a "medium daily dose for internal use: 2-4 g dried extract or 0.2-1.0 mg total hypericin in other forms of administration". It thus regards hypericin as the definitive active constituent. However, due to the subsequent availability of improved analytical and pharmacological experiments, we must now assume that the hypericins (proto-hypericins, pseudohypericins, cyclopseudohypericins, hypericin) are not the only important active constituents in Hypericum perforatum. As active substances also hyperoside, rutin, quercitin, hyperforin and chlorogenic acid must be considered. Proposed Mechanisms for the Antidepressant Action of Hypericum and Animal Models The following mechanisms are being discussed for the antidepressant action of St. John's wort: - Increase in the number of neurotransmitters (7) - Inhibition of type A monoamine oxidase (8) - Inhibition of catechol-O-methyltransferase (9) - Modulation of cytokine expressivity (10) - Hormonal effects (12) - Photodynamic effects, as in light therapy (13) The following animal experiments have been performed for the determination of the antidepressant action of St John's wort: - Exploration behaviour in unfamiliar environments (mice) (14) - Ethanol sleep period (mice) (14) - Action of reserpine (mice) (14) - Behaviour on the waterwheel (aggression of isolated male mice) (14) - Clonidine depression (gerbils) (14) - Forced swimming test (Porsolt) (12) The Amine Theory: Inhibition of MAO and COMT by Hypericum Extract Fractions One of the theories concerning the mechanism of action of Hypericum extracts is the socalled amine theory (15). It is based on the fact that in endogeneous depression an insufficient monoaminergic postsynaptic excitation-preferably by epinephrine and serotonin-is seen. The therapeutic effects of Hypericum extracts could be explained by either blocking the reuptake of serotonin and/or norepinephrine into the presynaptic nerve endings and/or by inhibiting the decomposition of epinephrine. By inhibiting the enzymes responsible for this process the levels of epinephrine could be increased. By the successive action of M A O and COMT, norepinephrine is transformed either to 3,4-



291 dihydroxymandelic acid or to vanillin-mandelic acid (Figure 2). The function of norepinephrine in the synapses, representing the contact place between the nerve cells and e.g. the sensory cells is demonstrated in Figure 3. Norepinephrine is located in the presynaptic vesicles and from there diffuses into the synaptic gap. At the postsynaptic membrane it induces an alteration of its permeability for ions which on their part modify the bioelectric polarisation of the membrane. By the enzymatic decomposition of the neurotransmitter, which also may consist of dopamine, serotonin, and G A B A (garnmaaminobutyric acid) the neurotransmitter effect is rapidly abolished and a repolarisation of the membrane takes place. Thiede (9) has investigated the inhibiting potential of several fractions of a Hypericum extract. The fractions 1-6 were obtained by extraction with different solvents resulting in the isolation of the components of the essential oil (Table I).

Table I. Solvents used for the preparation of extracts from 100 mg Hypericum perforatum and composition of the fractions obtained. Fractions obtained are characterized by the total amounts in mg and in % of the total amounts present in the original extract (9). Fraction 1: Solvent: petrol ethenether 1:1 yields 37 mg (2%) lipid-soluble components such as n-alkenes, pinene and other compounds of the essential oil Fraction 2: Solvent: ether yields 66 mg (3%) lipid soluble components Fraction 3: Solvent: ether.acetone = 8:2 yields 34 mg (2%) lipid-soluble components as fraction 1 Fraction 4: Solvent: acetone yields 47 mg (2%), little hypericin and flavonols Fraction 5: Solvent: methanohacetone (1:1) = yields 189 mg (8%) hypericin, little flavonols Fraction 6: solvent: methanol yields 1824 mg (83%) flavonoids, flavone glycosides, xanthones

By comparing the composition of the fractions with their ability to inhibit M A O and COMT, it becomes evident that St John's wort extract only inhibits M A O and COMT activity at relatively high concentrations (Table II). The COMT inhibition is mainly due to the flavonoids or flavone glycosides. The M A O inhibition, due to as yet unidentified constituents, can be observed in all high concentrations of St John's wort extract fractions. The M A O inhibition caused by isolated hypericin is considerably less than that of the total extract. Only at therapeutically irrelevant high doses of total extract (10" - 10 M) can an essential inhibition of M A O , but not of COMT, be seen. From these findings it can be concluded that the effect of the extract on norepinephrine metabolism is not only due to hypericin, and that inhibition of M A O and COMT is not the main mechanism by which Hypericum extracts exert their therapeutic effects (9). 3


A

292


HO

v 3,4-dlhydroxymandellc acid

HO^QV-CH-CH COOH«« 2

^ ' OH HO

o .

Aldehyde dehydrogenase (AD)

H-CH OH

aldehyde of 3,4-dihydroxymandellc acid ;ld

Aldehyde reductase (AR)

t

3,4-dihydroxyphenylethylglycol

HO

MAO

MAO

Q V - C H - C H £>H

HO

"

OH

HO

COMT

1

'-CM

HO-

H-CH2-i>Jn2 -NH 0

H jCO CO | 3

N

v

f

— c

norepinephrine COMT

CH-CH O H

HO

2

> f Cs\ OH f 3-methoxy-4-hydroxyAD phenylethylglycol MAO

^

'

OH

normetanephrlne (3-methoxy-noreplnephrine)

H-.CO

AR

HO

'

"~^0^~~

CH-COOH OH

3-methoxy-4-hydroxymandellc acid (vanlllin-mandellc acid)

Figure 2. Decomposition of norepinephrine (MAO = monoamine oxidase, COMT = catecholarnine-O-methyltransferase).



293

Figure 3. Metabolism of epinephrine in the synapses.


294 Table IL Inhibition (%) of MAO (M) and COMT (C) in vitro by extracts from Hypericum perforatum. For extracts and fractions, the mean mol mass of substance mixtures was taken as that of hypericin, i.e. 502 (9).


Concentration (M) Solvent Hypericin Hypericum extract Extract traction 1 Extractfraction2 Extract fraction 3 Extractfraction4 Extractfraction5 Extract fraction 6

io-

3

c

M 12 25 68 39 39 71 51 59 42

0 0

11 0 10 0 83 93

10^ M 0 2 27 16 0 29 4 5 5

io-

5

IO"

c

0 0 35 7 0 0 0 16 21

M 0 0 0 5 0 13 0 0 0

c

M 0 0 0 0 0 0 0 0 0

0 0 0 6 0 0 0 0 4

6

C

0 0 0 0 0 0 0 0 0

Results from experiments by Bladt and Wagner (8) on the M A O inhibition of hypericins and St John's wort fractions are essentially the same as those found by Thiede (9). Of the various St. John's wort constituents quercetin, luteolin and kaempherol, found in concentrations of about 10" M , demonstrated the highest inhibitory action (80-95%). Similar inhibition was shown by xanthones (70-90%) and hydroxyxanthones (60-80%). 1,3 Dihydroxyxanthone showed inhibition in concentrations of 10" M (70%) to 10* M (40%). The phloroglucin derivative, hyperforine, which occurs in St. John's wort in concentrations of about 3% showed only weak inhibition of less than 20% at a concentration of 10" M . At this same molar concentration, quercitrin was the only polar compound to demonstrate inhibition (65%) whilst the phenol carboxylic acids showed no effect. The total hypericin fraction only showed an inhibitory effect (70%) at a high concentration of 10" M . At 10 M both the total hypericin extract and pure hypericin showed less than 10% M A O inhibition, contrary to the 27% inhibition by the extract reported by others (Table II). These findings again establish that hypericins cannot be regarded as inhibitors of M A O . While the amine-theory can explain the mode of action of the classical antidepressants, it is insufficient to explain the therapeutic effect of Hypericum extracts. Especially the latency period of several weeks between drug application and antidepressant effect can not be explained on the basis of this theory. 5

3

5

3

4

Effect of Hypericum Extract on the Expression of Serotonin Receptors In addition to an increased availability of the neurotransmitter, i.e. by inhibition of M A O , the antidepressant effects of Hypericum extracts could be explained by a decrease in the number of serotonin receptors. Muller and Rossol (7) investigated the effect of Hypericum extract on the expression of serotonin receptors using a neuroblastoma cell line to establish a model for the regulation of neurotransmitters by immunologically active compounds such as cytokines. The cells were incubated with the Hypericum


295


extract LI 160 for 2,4,6,8 and 10 hours, then washed. Examination of the cells by means of fluorescence microscopy showed maximum reduction of the expression of the serotonin receptors after 6 hours of incubation. Also by diluted extract solutions a distinct reduction of receptor expression occured. From the LI160-induced reduction of the serotonin receptor availability, resulting in a decreased reuptake of the neurotransmitter into the cell, the authors conclude a possible mechanism of the antidepressant effect of the Hypericum extract. Contrary to the classical antidepressants, which bind to the receptor, it is suggested that neighboring epitopes of the serotonin receptors are occupied by the constituents of Hypericum, with subsequent masking of the receptors. The Macrophage Theory of Depression Besides direct effects on neurotransmitter metabolism, indirect effects mediated by immune modulators have been proposed for the antidepressant effects of Hypericum extract. The immune system and the nervous system are the only biological systems which are equipped with the ability of a memory. These systems are characterized by numerous regulatory interactions and biochemical correspondence. Important molecules are produced together by both systems, excreted and recognized as active substances such as endorphins, ACTH (adrenocorticotropic hormone), ADH (antidiuretic hormone) and others. Provided that the constituents of the Hypericum extract can not penetrate the blood-cerebrospinal fluid-barrier, their primary point of attack would be located outside of the central nervous system. The effect must then be mediated by CSF-penetrating substances. In this case effects on the cells of the immune system, such as lymphocytes and monocytes, could be expected. Taking into consideration these interactions between the immune system and the nervous system, the following mechanism could be supposed: stimulation of monocytes induces release of IL-6 (interleukin-6) which in turn influences, via the corticotropin releasing hormone and the lymphocytes, the release of ACTH. ACTH then induces the release of Cortisol which converts the normal emotional state into a depressive one (10). In his macrophage theory of depression, Smith (16) has suggested that in predisposed subjects, monocyte-interleukins are able to induce all symptoms of depressive states. Therefore, various specific or unspecific stimuli of monocuclear cells, such as infections, allergies, and estrogens, would induce an increase of interleukinelevels resulting in mental changes (Figure 4). Based on these considerations Thiele and coworkers (10) investigated the effect of Hypericum extract on the stimulated cytokine expression in vitro in a whole blood culture system. The assessment of the PHA(phytohemagglutinin)- and lipopolysaccharide-stimulated and unstimulated release of the cytokines interleukin IB, interleukin 6 and tumor-ncrose-factor-a (TNF-a) was performed with anticoagulated blood from 4 healthy and 4 depressive patients. Hypericum extract LI 160 significantly depressed the release of IL-6 stimulated by phytohemagglutinin and lipopolysaccharide by 92, 89, 90 and 97% in healthy persons and by 39, 95, 87 and 94% in depressive patients. In contrast to the stimulated release of IL-6, the release of IL-1B and TNF-a was influenced to a lesser extent. Furthermore, investigations of Muller and Rossol (7) have shown an increased expression of IL-1 on neurodermal cells, pointing out that also IL-1 directly takes part in the neuronal control.



296

stimulation of monocytes

Hypericum extract

I—I

IL-6

• CRH

1

i

lymphocytes

• ACTH

i Cortisol J

depression Figure 4. Modulation of cytokine expression by the Hypericum extract LI 160.


297


Central Effects of Hypericum Extracts, Being Mediated by the Dopaminergic System In vivo, Okpanyi and Weischer (14) were able to demonstrate a prolongation of an anaesthesia by ethanol, an increase of the spontaneous motility, a partial reserpin antagonism, and a suppression of the aggression behaviour of isolated male mice. However, these experiments have been critisized in so far as the Hypericum extract used had not been characterized phytochemically, the extract was applied in high doses, 50% propanediol was used as solvent, and hypericin as reference substance had not been applied by the oral route such as the Hypericum extract, but was given intraperitoneally. More recently a series of pharmacological trials were performed to confirm the assumption that Hypericum extracts exert a central effect and that these effects are mediated by the dopaminergic system. Investigations of Winterhoff and coworkers (11) have shown that Hypericum extract in mice induces an increase in body temperature. The increase is significant one hour after the application of 250 and 500 mg Hypericum extract per kg body weight. The temperature remains elevated above the initial temperature for 4 hours. These changes of body temperature indicate that oral Hypericum extract exerts central effects. These indications of a central effect of Hypericum were further investigated in other experimental approaches by Winterhoff and coworkers (12). A standard model for the assessment of the central effects of drugs is to measure the influence on the ketamine induced sleeping time. Pretreatment of female mice with 50, 200 and 500 mg/kg Hypericum extract results in a dose-related shortening of the sleeping time. The effect was significant with 200 mg extract and highly significant with 500 mg. A somewhat smaller effect was seen after pretreating the animals with concentrations of pure hypericin of 0.15,0.6 and 1.5 mg/kg. The mean duration of anaesthesia by the extract was shortened by 4.3%, 18.9% and 36.5%, by hypericin at concentrations corresponding to that of the extract by 2%, 9% and 22.4%. The same effect as by 500 mg Hypericum extract was seen with 20 mg/kg bupropione. Pretreatment of the animals with the unspecific dopamine receptor blocker haloperidol and with the specific D -receptor sulpiride completely abolished the effects of Hypericum extract and bupropione. A more specific method for testing the antidepressant effect of drugs is the forced swimming test according to Porsolt (17). The method is based on the observation that a rat, when forced to swim in a situation from which there is no escape, will become immobile making only those movements neccessary to keep its head above the water. Hypericum extract caused a reduction in immobility time which was significant at 125 mg/kg. Further increasing the concentration did not result in an stronger inhibition. Again 20 mg bupoprione proved to be active (12). From the fact that the anti-immobility effect of Hypericum extract observed during the forced swimming test and the Hypericum-induced decrease of the ketamine induced sleeping time are antagonized by the unspecific dopamine receptor blocker haloperidol as well as by the specific D -receptor sulpiride, it can be concluded that the dopaminergic system is involved in the activity of the Hypericum extract LI 160. These results are in good accordance with the findings of Winterhoff et al. (12) that prolactin levels are reduced after repeated application of Hypericum extract. This also reflects a dopaminergic effect. 2

2


298


Conclusions Summarizing the results of the various pharmacological investigations done so far the following conclusions can be made: 1. The inhibition of monoamine oxidase does not seem to be the main principle of the antidepressant effect of Hypericum extract. Based on the composition of the extract, the hypericins do not influence M A O activity at therapeutically used doses in a clinically relevant manner. Other constituents, such as theflavonoids,have a distinctly stronger effect on M A O . 2. Hypericum extract decreases the expresison of serotonin reseptors in the CNS. The constituents which are responsible for this effect have not been identified so far. 3. Besides direct effects on neurotransmitter metabolism, indirect effects mediated by immune modulators seem to play a role in the antidepressant effect of Hypericum extract. Thus, Hypericum extract significantly depressed the stimulated release of IL-6, which in turn influences, via the corticotropin releasing hormone and the lymphocytes, the release of A C T H . ACTH then induces the release of Cortisol, which converts the normal emotional state into a depressive one. 4. From the results of investigating the effects of Hypericum extract on the ketamine induced sleeping time and the forced swimming test it can be concluded that the dopaminergic system is involved in the activity of Hypericum extract. 5. Further investigations are necessary to identify the constituents of Hypericum which are responsible for its antidepressant effects. Literature cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13. 14. 15. 16. 17.

Kommission E; Monographie "Johanniskraut - Hyperici herba"; Bundesanzeiger Nr. 228, 5.12.1984. Wichtl, M. Medpharm Scientific Publ. Stuttgart 1994, pp. 273-275. Niesel, St.Inauguraldissertation 1992, Freie Universität Berlin. Harrer, G., Hübner, W. D., Podzuweit H. Nervenheilk., 1993, 12, 297-30. Johnson, D., Ksciuk, H., Woelk, H., Sauerwein-Giese, E., Frauendorf, A. Nervenheilk., 1993,12, 328-330. Vorbach, E. U., Hübner, W. D., Arnoldt, K., H. Nervenheilk., 1993, 12, 290-296. Müller, W. E. G., Rossol, R. Nervenheil., 1993, 12,357-358. Bladt, S., Wagner, H. Nervenheilk., 1993, 12,349-52. Thiede, H. M., Walper, A. Nervenheil., 1993, 12, 346-348. Thiele, B., Brink, I., Ploch, M. Nervenheilk., 1993, 12, 353-356. Winterhoff, H., Hambrügge, M., Vahlensieck, U. Nervenheilk., 1993, 12, 341-345. Winterhoff, H., Butterweck, V., Nahrstedt, A., Gumbinger, H. G., Schulz, V., Erping, S., Boßhammer, F., Wieligman, A. In: Loew, D., Rietbrock, N. (Eds.); Phytopharmaka in Forschung und klinischer Anwendung. Steinkopff Verlag Darmstadt 1995; pp. 38-56. Martinez, B., Kasper, S., Ruhrmann, S., Möller, H. J. Nervenheilk., 1993, 12, 302307. Okpanyi, S. N., Weischer, M. L. Arzneim-Forsch/Drug Res 1987, 37, 10-13. Kretschmar, R., Stille, G., Psychopharmaka. In: Estler, C.-J. (Ed.) Pharmakologie und Toxikologie. Schattauer Verlag Stuttgart, New York, 1995, p. 203. Smith, R. S. Med. Hypotheses 1991, 35, 298-306. Porsolt, R. D. Eur. J.Pharmacol.1978, 47, 379-391. In Phytomedicines of Europe; Lawson, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

Chemistry and Biology of Hypericum perforatum (St. John's Wort

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