Dietary Chemicals and Brain Function Graham A

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Journal & Proceedings of the Royal Society of New South Wales, Vol. 135, p. 57–71, 2003 ISSN 0035-9173/03/020057–15 $4.00/1

The 33rd Liversidge Lecture for the Royal Society of NSW Dietary Chemicals and Brain Function graham a. r. johnston

Abstract Phytochemicals in our diet may play a vital role in maintaining the brain’s chemical balance by influencing the function of receptors for the major inhibitory neurotransmitter GABA. The flavonoids apigenin and epigallocatechin gallate, found in chamomile and green tea respectively, influence the way in which GABA receptors are modulated by drugs such as diazepam. Resveratrol, a flavonoid-like polyphenol found in red wine, acts on a subtype of GABA receptors consistent with its action as a cognitive enhancer. Bilobalide from Ginkgo biloba, a herb used in cognitive therapy, also influences GABA receptors. α-Thujone, a terpenoid in the alcoholic beverage Absinthe, acts in a similar manner to bilobalide on GABA receptors. ( )-Borneol and other terpenoids from Valerian, a herb used to promote sleep, enhance the effects of GABA. The effects of these phytochemicals on GABA receptors are consistent with the overall actions of the beverages and herbal preparations that contain them, thus providing a rational basis for the use of these beverages and herbal preparations. These studies provide evidence that chemicals in our diet may influence brain function in a positive way. The chemical nature of these substances may lead to the development of new therapeutic agents for the treatment of anxiety, epilepsy, memory disorders and insomnia.

Keywords: Brain function, chemicals, diet, balance, dosage

THE BRAIN’S CHEMICAL over excited brain (as in Figure 1) that can be BALANCE manifested as anxiety, agitation, exhilaration, convulsions and death. On the other hand, an Two simple chemicals, glutamic acid and excess of inhibition over excitation can be man- GABA (Figure 1), are responsible for most of ifested by depression, anaesthesia, coma and the communication between nerve cells in the death. The particular manifestations of such brain. Indeed, at a very simple level, brain func- imbalances in the brain depend on what neu- tion may be thought of as a balance between ronal circuitry is involved. excitation mediated by glutamic acid and inhibition mediated by GABA. Ethanol is an example of a chemical that All nerve cells in the brain have receptors acts on both sides of the brain’s chemical bal- for glutamic acid and GABA. Some 40% of ance. The CNS depression that results from in- nerve cells release glutamic acid as an excita- gestion of ethanol is due to a reduction in excitatory neurotransmitter, while a diﬀerent 40% re- tion mediated by glutamic acid acting on a sub- lease GABA as an inhibitory neurotransmitter. type of glutamate receptors known as NMDA The balance between these two chemical trans- receptors and to an enhancement of inhibition mitters is vital to normal brain function. An mediated by GABA acting on GABAA recep- excess of excitation over inhibition results in an tors.

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H2N COOH H2N COOH

COOH GABA Inhibition ⇓ ((( Glutamic acid ((( (((( ((( Exitation (((( (((( ¢B ⇓ ((( (((( ¢ B ¢ B

Figure 1: The brain’s chemical balance between excitation mediated by the major excitatory neurotransmitter, glutamic acid, and inhibition mediated by the major inhibitory neurotransmitter, GABA.

GABA RECEPTORS oocytes from the South African frog, Xenopus laevis following injection of either DNA or RNA GABA (whose name is derived from the old cloned from human brain and coding for par- chemical name, γ-aminobutyric acid) acts on ticular GABA receptor protein subunits. These three main types of receptor to influence brain oocytes have the necessary cellular machinery to function. GABAA and GABAC receptors are make the human proteins and assemble them on fast acting receptors that belong to the group the surface of the oocytes as functional GABA of receptors called ligand-gated ion channels receptors. The oocytes are approximately one (LGICs) (Chebib and Johnston, 2000). GABA millimetre in diameter and readily penetrated acts as the ligand gating these receptors to by glass microelectrodes. Using 2-electrode open channels specific for chloride ions, allowing voltage clamp methodology, the effects of chem- these ions to flow rapidly into nerve cells mak- icals on the function of these GABA receptors ing them more negative and thus harder to ex- may be assessed in a convenient quantitative cite. GABAB receptors act more slowly, induc- manner. For example, using recombinant recep- ing metabolic changes in nerve cells and belong tor technology, the effects of anti-anxiety agents to the group of receptors called G-protein cou- such as diazepam (Valium) on GABA recep- pled receptors (GPCRs) (Bowery et al. 1997). tors can be easily shown to be restricted to a specific sub-group of GABAA receptors. The The study of GABA receptors has been rev- technology is not restricted to the study of pure olutionised by the introduction of recombinant chemicals – relatively crude mixtures of chem- receptor technology whereby receptors cloned icals can be studied, for example to follow the from human brain are expressed in cells that do purification of chemicals acting on GABA re- not normally express such receptors (Barnard et ceptors from extracts of herbal products. al. 1998). The recombinant receptors so formed may be studied in relative isolation using stan- dard electrophysiological methodology. Since FLAVONOIDS AND TERPENOIDS all GABA receptors are made up of protein subunits, recombinant receptors of known subunit Flavonoids are polyphenolic chemicals widely composition may be studied. distributed in the plant kingdom particularly The most common way to study recombi- in flowering plants. Flavonoids are responsi- nant GABA receptors is to express them in ble for many of the brilliant colours of fruits

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and vegetables and are important constituents van, epigallocatechin gallate (Figure 2). of red wine, green tea and many herbal prepa- Terpenoids are also widespread in plants, es- rations (Aherne and O’Brien, 2002). Chemi- pecially in what are known as essential oils that cally, flavonoids are C15 compounds based on can be extracted from plants and have a wide the chromane ring structure. Flavonoids have range of uses from perfume constituents to paint been studied extensively as anti-oxidants and thinners. Terpenoids are oxygenated products oestrogens (Collins-Burow et al. 2000). Many formally derived from C5 isoprene units and are of them show anti-cancer and anti-viral proper- classified by the number of C5 units in their ties (Le Marchand, 2002). structure. Thus monoterpenoids have 2xC5 There is an extensive literature on the effects units, sesquiterpenoids 3xC5 units, diterpenoids of flavonoids on GABA receptors (for a recent 4xC5 units and triterpenoids 6xC5 units. In review see Marder and Paladini (2002), dating the present context of actions on GABA recep- from the discovery of some plant derived isofla- tors, the following terpenoids are of interest: vans in bovine urine that inhibited the bind- α-thujone, ( )-borneol, bilobalide and picrotox- ing of diazepam to brain membranes (Luk et inin (Figure 3). Picrotoxinin is widely used ex- al. 1983). In the present context of actions on perimentally as a non-competitive antagonist of GABA receptors, the following flavonoids are GABAA and GABAC receptors, however, its of interest: the flavone apigenin; the isoflavone convulsant action restricts its therapeutic use genistein; the flavanone naringenin; and the fla- (Chebib and Johnston, 2000).

OH HO O

HO O

OH O OH

OH O Genistein, an isoﬂavone found in soy products, including tofu. Apigenin, a ﬂavone found in chamomile tea and related beverages. OH

OH HO O OH HO O

OH OH O OH O

OH Naringenin, a ﬂavone found in grapefruit. OH (-)-Epigallocatechin Gallate, a ﬂavan found in green tea.

Figure 2: Some representative ﬂavonoids

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CH H C 3 CH3 3 H3C CH3 CH3 O

α-Thujone, a monoterpene ( )-Borneol, a monoterpene from Artemisia absinthium from Valerian oﬃcinalis

O O O O H H OH HO O O O OH O O

O O H

Bilobalide, a sesquiterpenoid Picrotoxinin, a sesquiterpenoid from from Ginkgo biloba Anamirta cocculus

Figure 3: Some terpenoids that inﬂuence GABA receptors

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APIGENIN FROM CHAMOMILE bell in our research group investigated the ac- TEA tion of apigenin on recombinant GABA receptors. She used electrophysiological recordings The lead compound for our investigations was from Xenopus laevis oocytes injected with re- apigenin (Figure 2), a flavonoid with a known combinant human RNA for the most common anti-anxiety action found in chamomile tea. subtype of GABAA receptor (α1β2γ2L) in the Chamomile tea is used widely to treat anxi- brain. The actions of GABA at these receptors ety and insomnia. Current therapeutic drugs are enhanced by a variety of modulators includ- used for the treatment of anxiety and insom- ing barbiturates, benzodiazepines, ethanol, and nia such as the benzodiazepines Valium and neuroactive steroids. Serepax act at GABAA receptors in the brain, She showed the action of apigenin on the increasing chloride flow into neurones, result- GABAA receptor is more complex than sug- ing in decreased neural activity. There were gested by earlier studies. The effects of api- divergent reports of the effects of apigenin on genin were biphasic dependent on the dose used. GABAA receptors. Viola et al. (1995) con- Moderate doses of apigenin inhibited the ac- cluded that apigenin is a benzodiazepine ago- tions of GABA, diazepam and the steroid al- nist, like diazepam. However, Avallone et al. lopregnanalone, whereas low apigenin concen- (2000) concluded that apigenin was a benzodi- trations enhanced the effects of diazepam only azepine inverse agonist (the exact opposite of (Figure 4). These effects are unlikely to be due diazepam). to a simple action at the benzodiazepine site, Viola et al. (1995) based their conclusion suggesting a new site on the GABAA receptor. that apigenin is a benzodiazepine agonist on While the inhibitory actions of apigenin at the ability of apigenin to displace the bind- moderate doses were consistent with it acting ing of radiolabelled benzodiazepines from rat as a benzodiazepine inverse agonist, the abil- brain membranes, coupled with benzodiazepine- ity of apigenin to enhance the enhancing action like effects in a rodent model of anxiety. How- of diazepam was novel. At low doses, apigenin ever, binding studies do not reliably distinguish had no direct effect on the action of GABA on between agonists, antagonists and inverse ag- these recombinant receptors. The presence of onists. Indeed, on the basis of similar bind- diazepam was necessary in order to observe the ing studies, Dekermendjian et al. (1999) con- enhancing effects of apigenin. Thus apigenin cluded that apigenin was a benzodiazepine an- appeared to be modulating the action of a mod- tagonist (that is, it binds to the benzodiazepine ulator, an action not previously described in site, blocking the binding of benzodiazepine ag- the pharmacological literature. Apigenin might onists and inverse agonists, without having any be described as a second order modulator that effect on GABA responses itself). Avallone et influences the modulatory action of diazepam al. (2000) used electrophysiological recordings as a first order modulator on the activation of from rat neurones. This allowed a more di- GABAA receptors. rect investigation of the activity of apigenin and The second order modulation of GABAA showed that apigenin inhibited currents due to receptors by apigenin requires the presence of GABA, an effect which was blocked by the ben- GABA and a first order modulator acting at zodiazepine antagonist, flumazenil. This fits a benzodiazepine site. The sedative and anx- the profile of a benzodiazepine inverse agonist. iolytic actions of apigenin observed in rodents However, Avallone et al. (2000) did find some (Avallone et al. 2000, Viola et al. 1995) can be behavioural effects of apigenin which could be interpreted on the basis of apigenin potentiating consistent with an action as a benzodiazepine the action of endogenous benzodiazepine-like agonist. agents in the brain. Evidence for the physiolog- As part of her PhD studies, Erica Camp- ically relevant presence of such agents, termed

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endozepines, has come from the discovery of a of apigenin, showed only the GABAA antago- mutant GABAA receptor in childhood absence nist action of apigenin. In addition, a dihydro epilepsy and febrile seizures that has diminished derivative of apigenin, naringenin (Figure 2), a sensitivity to benzodiazepines with little other flavanone found in grapefruit juice and other cit- alteration in GABAA receptor function (Wal- rus products, also showed only the GABAA an- lace et al. 2001). tagonist action of apigenin. Thus, the second Genistein (Figure 2), an isoflavone found order modulatory action of apigenin is struc- in soy products, did not show the biphasic ef- turally specific. fects of apigenin. Genistein, a structural isomer

2 Enhancement

onse 1.5 GABA resp M µ 5

+ 1 alone) diazepam of diazepam GABA 0.5 M M µ ortion µ (1=3 0=5 Prop 0 Inhibition

−0.5 0.01 0.1 1 10 100 Apigen Concentration (µM)

Figure 4: Eﬀects of apigenin on the enhancement by diazepam of the action of GABA on recombinant GABAA receptors (Campbell et al. 1999).

EPIGALLOCATECHIN GALLATE shown to contribute to these effects and, in ad- FROM GREEN TEA dition, has been shown to have neuroprotective properties. Epigallocatechin gallate (Figure 2, EGCG) is Erica Campbell investigated the actions the most abundant flavan in green tea (Camel- of EGCG on recombinant GABA receptors lia sinensis). It is found in all teas made from (Campbell et al. 1999). She found that it shared C. sinensis but not in many other food products the same biphasic action of apigenin, enhancing (Arts et al. 2000b). Green tea is known to have the action of diazepam at low concentrations many beneficial effects, including prevention of and inhibiting at higher concentrations. In both cancer, lowering of blood pressure and lipids, the enhancement and inhibition phases, EGCG and acting as an antioxidant. EGCG has been was at least 10 times more potent than apigenin.

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( )-Catechin and (-)-epicatechin, the most diazepines. The possibilities of interactions be- abundant flavans in nature, being found in tween benzodiazepine medication and the con- many foods (Arts et al. 2000a, Arts et al. sumption of chamomile and green tea need to be 2000b), did not influence recombinant GABA considered, particularly as chamomile tea may receptors, showing that the basic flavan ring be used as a home remedy for those conditions structure is not sufficient for either of the for which benzodiazepines are frequently pre- actions of EGCG observed on recombinant scribed. GABAA receptors. The biphasic actions of apigenin and EGCG emphasise the importance of dose in drug ac- RESVERATROL FROM RED WINE tion. Our experiments show that low doses of apigenin and EGCG can enhance the activation The relatively low incidence of coronary heart of GABA receptors under the right conditions diseases in France, despite intake of a high- and thus could produce sedation and relief of fat diet, – the “French Paradox” – has been anxiety. On the other hand, higher doses have attributed to the consumption of red wine the opposite effect and thus are likely to pro- containing high levels of polyphenolic com- duce stimulation. pounds (Mojzisova and Kuchta 2001, Sun et al. The second order modulatory action of api- 2002). Resveratrol (3,4’,5-trihydroxystilbene, genin and EGCG might have therapeutic possi- Figure 5) is one of the most interesting polyphe- bilities. Low doses of these substances could nolic compounds found in red wine. It has been reduce the therapeutic dose of diazepam and shown to have estrogenic (Turner et al. 1999) related benzodiazepines, while higher doses and neuroprotective effects (Bastianetto et al. might reduce the effectiveness of such benzo- 2000).

Resveratrol, a stilbene found in red wine from Vitis vinifera OH

Figure 5: Structure of Resveratrol

In view of the structural similarities be- competitively inhibited the effects of GABA tween resveratrol and apigenin, we investigated (1 µM) at GABAC receptors with an IC50 of its effects on recombinant GABA receptors ex- 72 µM, while having no significant effect at pressed in oocytes. To our surprise, resvera- doses up to 100 µM on the effects of GABA trol showed little action on GABAA receptors (40 µM) at GABAA receptors. This is the first but was a GABAC receptor antagonist (Camp- report of a non-competitive antagonist show- bell and Johnston, 2003). Resveratrol non- ing some selectivity for GABAC over GABAA

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receptors, the widely used non-competitive an- effectiveness of GABA acting on GABAA recep- tagonist picrotoxinin being some 30 times more tors and there is evidence that moderate con- potent at GABAA than at GABAC receptors sumption of alcoholic beverages is beneficial to (Chebib and Johnston, 2000). health. However, other substances in these bev- We have a patent on the use of GABAC re- erages, such as resveratrol, are likely to con- ceptor antagonists to enhance cognitive activ- tribute to the overall beneficial effects. Re- ity and stimulate memory capacity (Johnston et cently it has been reported by Aoshima and col- al. 1998). Thus it was interesting to note that leagues (Hossain et al. 2002) that the fragrance resveratrol has also been patented for the treat- of whiskey is able to enhance the effectiveness ment of mild cognitive impairment (Wurtman of GABA acting on GABAA receptors. Several and Lee, 2002) based on its ability to increase components of the fragrance showed this prop- the expression of soluble amyloid precursor pro- erty, the most potent being ethyl phenylpropi- tein. onate, which was shown to have an anticonvul- Using resveratrol as a lead compound, we sant action in mice on inhalation. are examining structural analogues to see if we Enhancement of GABA action was also can develop more potent and selective com- found in extracts of other alcoholic drinks such pounds acting on GABAC receptors. Recently as wine, sake, brandy and sochu. Hossain we discovered a range of very promising activ- et al. (2002) noted “Although these fragrant ities, including the ability to enhance GABAC components are present in alcoholic drinks at receptor activity, in a series of compounds syn- low concentrations (extremely small quantities thesized in the 1970s by David Collins and col- compared with ethanol), they may also modu- leagues in Veterinary Physiology at The Univer- late the mood or consciousness of the human sity of Sydney as antiestrogenic and antifertility through the potentiation of the GABAA recep- agents (Collins et al. 1971). tor response”. Aoshima and colleagues have There is great interest in drugs to treat shown that various perfume constituents and memory impairment in disorders such as aromatherapy agents potentiate GABAA recep- Alzheimer’s disease and schizophrenia. The use tors (Aoshima and Hamamoto 1999, Aoshima of such “Smart Drugs” in healthy people to in- et al. 2001). crease their cognitive ability raises a variety of Clearly there are many interesting and inno- ethical, legal and social issues (Rose 2002). vative ways to explore the possibilities of influencing cognitive function. Resveratrol and related stilbenoids are found in a variety of plants and herbs. Major dietary sources include grapes, wine, peanuts and BILOBALIDE FROM GINKGO soy (Burns et al. 2002). These compounds are BILOBA also found in Itadori tea which has long been used in Japan and China as a traditional rem- Extracts of Ginkgo biloba leaves are widely em- edy for heart disease and stroke. For people ployed as herbal medicines to treat symptoms who do not wish to consume alcohol, Itadori associated with mild-to-moderate dementia, im- tea may be a substituent for red wine as a di- pairment of other cognitive functions associated etary source of resveratrol (Burns et al. 2002). with ageing and senility and related neurosen- For those who prefer white wine to red, French sory problems (Diamond et al. 2000). A study winemakers have created a Chardonnay, called has indicated that the cognition-enhancing ef- Paradoxe Blanc, that is enriched in polyphenols fects of the Ginkgo leaf extracts may be partly and has been shown to be effective in reducing mediated by bilobalide via GABA receptors oxidative stress in diabetic rats (Landrault et (Sasaki et al. 1999b). Enhanced hippocampal al. 2003). pyramidal neuronal excitability has been shown As noted above, ethanol itself enhances the to correlate with learning and memory (Power

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et al. 1997). Bilobalide (Figure 3), a sesquiter- potent insecticide (Ahn et al. 1997), an action penoid isolated from Ginkgo biloba leaves, has likely to be due to blockade of insect GABA re- been shown to enhance this excitability in rat ceptors. hippocampal slices, an action proposed to in- volve blockade of GABAergic neurotransmis- sion (Sasaki et al. 1999b). THUJONE FROM ABSINTHE In collaboration with Sasaki and colleagues, Absinthe was the favoured drink of artists and Shelly Huang and Rujee Duke in our research writers in Paris at the end of the 19th cen- group have shown that bilobalide is a potent an- tury. The emerald green liqueur made famous tagonist of the action of GABA on recombinant by Van Gogh, Toulouse-Lautrec and their col- GABAA and GABAC receptors (Huang et al. leagues was banned in France and most other 2003). Bilobalide was only marginally less po- countries by 1915 due to its ability to cause tent than picrotoxinin in these actions but there convulsions, hallucinations and psychotic dis- were subtle differences between the actions of turbances. bilobalide and picrotoxinin. These findings The toxic component of absinthe has been strongly support the proposal by Sasaki and col- identified as the monoterpenoid α-thujone (Fig- leagues (Sasaki et al. 1999b) that the observed ure 3). It is also the active ingredient of worm- enhanced neuronal excitability in hippocampal wood oil and some other herbal medicines and slices was due to its blockade of GABAergic neu- is reported to have antinociceptive, insecticidal, rotransmission. and anthelmintic activity. Hold et al. (2000) Bilobalide and picrotoxinin share common showed that α-thujone acted like picrotoxinin structural features, including a hydrophilic cage as a GABAA receptor non-competitive antago- and lipophilic side chain. However, bilobalide nist. They showed that α-thujone was rapidly and picrotoxinin have opposite actions upon metabolised to less active metabolites and con- systemic administration to animals. Bilobalide cluded that “α-thujone in absinthe and herbal is an anticonvulsant (Sasaki et al. 1999a,b) medicines is a rapid-acting and readily detox- whereas picrotoxinin is a potent convulsant ified modulator of the GABA-gated chloride (Jarboe et al. 1968). There are, however, only channel”. minor differences in their activities at recom- Matthew Roper in our research group has binant GABAA and GABAC receptors. Bilob- shown that α-thujone is a non-competitive in- alide has been shown to increase GABA lev- hibitor of the action of GABA on recombinant els in the hippocampus and cerebral cortex of α1β2γ2L GABAA and ρ1 GABAC receptors ex- mice (Sasaki et al. 1999a). This increase may pressed in oocytes. Like picrotoxinin, α-thujone override the GABAA antagonist action of bilob- was about 30 times more potent at GABAA alide that would be expected to produce convul- than at GABAC receptors. Furthermore, site- sions and result in the overall anticonvulsant directed mutagenesis studies showed that muta- action. Nonetheless, the induction of epilep- tions in the second membrane-spanning region tic seizures by Ginkgo extracts has been noted of the wildtype GABAC receptors influenced the in rare cases (Granger, 2001). The anticonvul- potency of α-thujone and picrotoxinin in a simi- sant/convulsant actions of bilobalide need fur- lar manner indicating that both convulsants in- ther investigation and may provide vital clues as teract with the same amino acid residues on the to the safe use of Ginkgo extracts in the treat- GABAC receptor. ment of mild cognitive deficits. Many plant-derived essential oils, such as Ginkgo leaves were used traditionally in wormwood, have been known for over a cen- Japan to protect books against harmful worms tury to have convulsant properties. Burkhard and insects before the introduction of modern et al. (1999) reported on case studies of plant- insecticides. Like picrotoxinin, bilobalide is a related toxic seizures related to use of these oils

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for therapeutic purposes. They noted that “the also contain various flavonoids, alkaloids, tan- literature shows essential oils of 11 plants to be nins and amino acids. powerful convulsants (eucalyptus, fennel, hys- Renee Granger in our research group ob- sop, pennyroyal, rosemary, sage, savin, tansy, tained 2 kg of the dried underground parts thuja, turpentine, and wormwood) due to their of Valeriana officinalis from Newton’s Herbal content of highly reactive monoterpene ketones, Pharmacy in Sydney. She extracted this such as camphor, pinocamphone, thujone, cine- with hexane, ethyl acetate, methanol and ole, pulegone, sabinyl acetate, and fenchone.” methanol:water (1:5), and fractionated the ex- They went on to state “Nowadays the wide use tracts using silica gel chromatography. This of these compounds in certain unconventional procedure produced more than 450 fractions, medicines makes this severe complication again which were assessed using thin layer chromatog- possible”. raphy and functional studies on recombinant re- Absinthe is now available in a less potent ceptors many fractions influenced GABA action form that contains less than 10 parts per mil- on GABAA and GABAC receptors. lion of α-thujone, whereas traditional absinthe Dried valerian root normally contains 0.3– contained more than 250 parts per million. 0.8% volatile oil, including borneol, valerenic acid, valeranone and kessyl glycol . These essen- BORNEOL FROM VALERIAN tial oil fractions proved very difficult to purify, so pure compounds were purchased and tested Valerian (Valeriana officinalis) is a medicinal on recombinant receptors. This produced a very plant used widely throughout the world. Ex- surprising result. tracts of the dried underground parts of the ( )-Borneol, the natural enantiomer found plant are used to relieve anxiety, restlessness in Valerian, produced a 12 fold enhancement and nervous sleep disorders. There is evidence of the action of 10 µM GABA on recombinant of its use by the ancient Greeks, Romans and GABAA receptors under conditions whereby di- Chinese for healing purposes. Early herbalists azepam would give at best a 2 fold enhancement and physicians such as Hippocrates, Galen and (Figure 6; Granger et al. 2002). While relatively Culpeper noted the sedative and digestive prop- high concentrations of ( )-borneol were needed erties of valerian, advocating its use as a mus- (EC50 400 µM), this degree of enhancement is cle relaxant, diuretic, expectorant and wound unprecedented. healer (Plushner, 2000). Today there are over Under these conditions, (-)-borneol pro- 400 commercially available products containing duced a 4 fold enhancement (EC50 450 µM), valerian in Germany, more than 80 in the United isoborneol a 7 fold enhancement (EC50 Kingdom and more than 30 available in Aus- 680 µM), while the structurally related tralia (Houghton, 1999; Shohet et al. 2001). monoterpenes camphor, bornyl acetate and p- Valerian extracts may be considered to be cymene each produced an approximately 3x a “herbal Valium”, given that they have a potentiation (with EC50’s around 300 µM). benzodiazepine-like action reducing the latency While many general anaesthetics, barbitu- of sleep onset and increasing the depth of sleep rates and benzodiazepine are known to pro- and the perception of well-being. These extracts duce up to 2 fold enhancement of the re- contain a large number of constituents, many of sponse of GABAA receptors to GABA (Belelli which are thought to be active at GABA re- et al. 1999b), the general anaesthetic etomidate ceptors. Compounds that have been identified is known to cause much larger enhancements, include acids (valerenic acid and isovalerenic particularly at mutated GABA receptors, e.g. acid), ketones (valeranone), alcohols (valerenol, etomidate produces a 10 fold enhancement of maaliol), aldehydes (valerenal) and valepotri- GABA responses at GABAC receptors where ates (valtrate, isovaltrate). Valerian extracts the wild type isoleucine residue at position 307

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had been mutated to a serine (Belelli et al. Borneol is a common constituent of the essential 1999a). oil component of many plants and thus a com- ( )-Borneol represents an intriguing struc- ponent of many herbal preparations. On the tural lead for the development of a new class of basis of our studies, ( )-borneol would be ex- therapeutic agents acting on GABA receptors. pected to have antianxiety, anticonvulsant and Puriﬁed ( )-borneol has been used for medicinal sedative properties. purposes in China and Japan (Hattori, 2000).

( )-Borneol

(-)-Borneol

10 onse resp of GABA M µ ortion 5 10 to Prop

0 −2 −1 0 1 2 3 4 Log Dose (µM)

Figure 6: Dose-response curve for the potentiation of the response to 10 µM GABA by ( )- and (-)-borneol at recombinant GABAA receptors (Granger et al. 2002).

NATURAL VERSUS SYNTHETIC Australia, using high throughput technology to screen extracts of natural products for desired Natural products derived from plants provided biological activities. the ﬁrst medicines. These were complex mix- Herbal preparations are by their nature mixtures of chemicals. The active principles in tures of chemicals. It is a basic tenet of herbal these mixtures were isolated and developed as medicines that the whole is more than the sum single chemical entities to use as drugs and of the parts. The various chemicals in herbal from which to develop new therapeutic agents. preparations are considered to act together in The development of aspirin from the salicylates a synergistic way to eﬀect treatment of partic- found in Willow bark is a classic example of ular disorders. This is in direct contrast to the this. Natural products continue to be an im- “magic bullet” approach of single chemical en- portant source of new drugs. There are sophisti- tity drugs designed to hit a particular target in a cated laboratories in many countries, including highly selective manner. Both approaches have

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their role in promoting health and well-being. genin (Figure 2), that inhibit the p450 enzyme There is a widespread belief on the part of CYP3A4 resulting in higher bioavailability of the general public that natural substances are drugs with a high firstpass metabolism (Fuhr inherently superior to synthetic substances with 1998). While there may be a place for grape- regard to efficacy and safety in matters related fruit juice as a drug-sparing agent, more re- to human health. This question has been ad- search is needed and drugs possibly influenced dressed recently by a task force of the Inter- by the consumption of grapefruit juice should national Union of Pure and Applied Chemistry be appropriately labelled. (Topliss et al. 2002). A comparison of the char- acteristics of natural and synthetic substances CONCLUSIONS used in a variety of therapeutic drugs, herbal medicines, vitamins and nutrients shows a sim- GABA receptors in our brain are susceptible to ilar range of favorable and unfavorable effects. a wide variety of chemicals consumed in the It was apparent that molecular structure and diet. Our GABA receptors exist in a milieu dose determine the effect of chemicals on hu- of substances that influences their function, of- man health, not whether they are of natural or ten in opposing ways. The balance between synthetic origin. the effects of these substances will determine Natural chemicals such as many flavonoids at any particular point in time how the recep- have been consumed in our diet for countless tors respond to GABA, their natural neuro- generations. This suggests that they would transmitter. This review has summarised the be unlikely to have serious adverse effects se- effects of some substances found in four bever- vere enough to prevent their use as therapeutic ages (chamomile and green tea, red wine, ab- agents. However, it is likely that the overall bal- sinthe) and two herbal preparations (Gingko ance of flavonoids and related natural chemicals and Valerian) that have significant actions on in our diet is of vital importance, given the ex- recombinant GABA receptors consistent with amples in this review of such chemicals having the overall actions of the beverages and herbal opposing actions on GABA receptors. Recom- preparations. The chemical nature of these sub- binant receptor technology offers the means to stances may lead to the development of new assess the overall effects of complex mixtures of therapeutic agents for the treatment of anxi- chemicals on the functioning of key receptors. ety, epilepsy, memory disorders, and insomnia. Such technology is expected to play an increas- Does the concept of dietary substances influenc- ingly important role in the quality control of ing brain function indicate that we have entered herbal preparations and “functional foods”. an era of neuraceuticals? Herbal preparations can have significant in- These studies provide a chemical basis for teractions with therapeutic drugs, for example some of the effects that these beverages and by altering the metabolism of these drugs and herbal preparations have on brain function, and thus influencing their potency and duration of may lead to rational improvements in their qual- action (Izzo and Ernst 2001). It is important ity control and use, especially in combination that health care professionals ask their patients with other agents known to influence GABA re- about their use of herbal products and consider ceptors, such as alcohol, anaesthetics and ben- the possibility of herb-drug interactions. Food- zodiazepines. drug interactions are also important (Sorensen 2002) as many naturally occurring substances ACKNOWLEDGEMENTS influence the cytochrome p450 enzymes that play such an important role in drug metabolism. It is a pleasure to gratefully acknowledge the Grapefruit juice is a classic example. It con- collaboration in these studies of Erica Camp- tains substances, including the flavonoid narin- bell, Mary Chebib, Rujee Duke, Renee Granger,

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Belinda Hall, Jane Hanrahan, Shelly Huang, Barnard, E.A., Skolnick, P., Olsen, R.W., Ken Mewett, Matthew Roper and Hue Tran. Möhler, H., Sieghart, W., Biggio, G., Appreciation is also due to Gary Cutting, Braestrup, C., Bateson, A.N. and Langer, George Uhl and Paul Whiting for the supply of S.Z. 1998. International Union of Pharmacol- cloned GABA receptors and to the Australian ogy - XV - Subtypes of γ-aminobutyric acidA National Health and Medical Research Council receptors - classification on the basis of sub- for financial support. unit structure and receptor function. Phar- macological Reviews, 50, 291–313. REFERENCES Bastianetto, S., Zheng, W.H. and Quirion, R. 2000. Neuroprotective abilities of resveratrol Aherne, S.A. and O’Brien, N.M. 2002. Di- and other red wine constituents against nitric etary flavonols: Chemistry, food content, and oxide-related toxicity in cultured hippocam- metabolism. Nutrition, 18, 75–81. pal neurons. British Journal of Pharmacol- ogy Ahn, Y.J., Kwon, M., Park, H.M. and Han, , 131, 711–720. C.K. 1997. Potent insecticidal activity of Belelli, D., Pau, D., Cabras, G., Peters, J.A. Ginkgo biloba derived trilactone terpenes and Lambert, J.J. 1999a. A single amino against Nilaparvata lugens. Phytochemicals acid confers barbiturate sensitivity upon the for pest control. American Chemical Society GABA ρ(1) receptor. British Journal of Symposium Series, 658, 90–105. Pharmacology, 127, 601–604. Aoshima, H. and Hamamoto, K. 1999. Poten- Belelli, D., Pistis, M., Peters, J.A. and Lambert, tiation of GABA(A) receptors expressed in J.J. 1999b. The interaction of general anaes- Xenopus oocytes by perfume and phytoncid. thetics and neurosteroids with GABA(A) and Bioscience Biotechnology and Biochemistry, glycine receptors. Neurochemistry Interna- 63, 743–748. tional, 34, 447–452. Aoshima, H., Hossain, S.J., Hamamoto, K., Bowery, N.G., Kerr, D.I.B. and Ong, J. (997. Yokoyama, T., Yamada, M. and Shingai, R. GABA-B receptors - role and therapeutic im- 2001. Kinetic analyses of alcohol-induced po- plications in CNS disorders. CNS Drugs, 7, tentiation of the response of GABA(A) recep- 167–175. tors composed of alpha(1) and beta(1) sub- Burkhard, P.R., Burkhardt, K., Haenggeli, C.A. units. Journal of Biochemistry, 130, 703– and Landis, T. 1999. Plant-induced seizures: 709. reappearance of an old problem. Journal of Arts, I.C., van de Putte, B. and Hollman, P.C. Neurology, 246, 667–670. 2000a. Catechin contents of foods commonly Burns, J., Yokota, T., Ashihara, H., Lean, consumed in The Netherlands. 1. Fruits, M.E.J. and Crozier, A. 2002. Plant foods and vegetables, staple foods, and processed foods. herbal sources of resveratrol. Journal of Agri- Journal of Agricultural and Food Chemistry, cultural and Food Chemistry, 50, 3337–3340. 48, 1746–51. Campbell E.L., Chebib M. and Johnston Arts, I.C., van De Putte, B. and Hollman, P.C. G.A.R. 1999, The flavonoid epigallocatechin 2000b. Catechin contents of foods commonly gallate blocks steroid, benzodiazepine and consumed in The Netherlands. 2. Tea, wine, GABA actions at the GABA-A receptor more fruit juices, and chocolate milk. Journal of potently than apigenin. Proceedings of the Agricultural and Food Chemistry, 48, 1752–7. Australasian Society of Clinical and Experi- Avallone, R., Zanoli, P., Puia, G., Kleinschnitz, mental Pharmacology and Toxicology, 6, 109 M., Schreier, P. and Baraldi, M. 2000. Phar- Campbell, E.L. and Johnston G.A.R. 2003. macological profile of apigenin, a flavonoid Resveratrol is a GABAC receptor antagonist. isolated from Matricaria chamomilla. Bio- Proceedings of the Australian Neuroscience chemical Pharmacology, 59, 1387–1394. Society, 14, P156

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