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THYMOL C10H14O
PRODUCT IDENTIFICATION
CAS NO.	89-83-8
EINECS NO.	201-944-8
FORMULA	C10H14O
MOL WT.	150.22
H.S. CODE	2907.19
TOXICITY	Oral rat LD50: 980 mg/kg
SYNONYMS	p-Cymen-3-ol; Thyme camphor; m-Thymol;
3-Hydroxy-p-cymene; 3-Methyl-6-isopropylphenol; 6-Isopropyl-m-cresol; Timol; 6-Isopropyl-3-methylphenol; Isopropyl cresol; 2-isopropyl-5-methyl phenol; Thymic acid; 1-Hydroxy-5-methyl-2-isopropylbenzene; 1-Methyl-3-hydroxy-4-isopropylbenzene; 3-p-Cymenol; 3-Hydroxy-1-methyl-4-isopropylbenzene; 5-Methyl-2-isopropyl-1-phenol; 5-Methyl-2-(1-methylethyl)phenol; 6-Isopropyl-m-cresol;  2-Isopropyl-5-methylphenol;
DERIVATION
CLASSIFICATION
PHYSICAL AND CHEMICAL PROPERTIES
PHYSICAL STATE	white crystal
MELTING POINT	48 - 51 C
BOILING POINT	233 C
SPECIFIC GRAVITY	0.965
SOLUBILITY IN WATER	Very slightly (soluble in paraffin oil; alcohol)
pH
VAPOR DENSITY	5.2
AUTOIGNITION
REFRACTIVE INDEX	Health: 3 Flammability: 1 Reactivity: 0
NFPA RATINGS
FLASH POINT	102 C
STABILITY	Stable under normal conditions.
GENERAL DESCRIPTION & APPLICATIONS
English:Thyme Scientific term:Thymus vulgaris The musk grass Fen(Thymol) The musk grass extracts(Thyme Extract) Musk grass Fen. Thymopoietin The thyme brain[Fen]; Musk grass Fen thyme camphor Nickname:Musk grass Section:Lips form section Classification:Often green and small bush(get herbage for many years) At first habitat:Mediterranean littoral The stub is high:Straighten a type 20-40 cm, crawl a type 10 or so Stub:30-40 Bloom a period:3-6 months in Taiwan, foreign 5-7 months Make use of part:Leaf, flower, caulis Flavor characteristic:Have the sweet smell ingredient of the musk grass Fen, have elegant and heavy spirit. Usage:The cuisine, health, hairdressing, tea drinks, ethereal oil, flower arrangement, horticulture, craft Suggest effect:Promote digest, disinfect, strong, disinfect, antisepsis Thymol (Thyme Camphor): a white crystals with camphor like odor, is a phenolic compound obtained naturally from thyme oil (or other volatile oils) or prepared synthetically. It is very slightly soluble in water; soluble in paraffin oil and alcohol. It is used as a stabilizer in pharmaceutical and as a topical antiseptic, antibacterial, and anti-fungal agent. It is also used as a flavoring agent for drugs (camphor, herbal, wintergreen,disinfectants, origanum )

Have the herbs of the stimulating smell, also make the food seasoning, bee good thyme, for hundreds of years, the cc mile thyme Mi always famous, in ancient Greece sanctuary, the thyme has ever used to Xun joss-stick, it is produced in an European south at first, region in Mediterranean, Asia minor with do a leaf Central Asia brown green, crush pieces aroma.The flavor mightiness, mordacity, its ethereal oil is in common use to make in the spice and toothpaste, having antisepsis and anaesthetizing a function, also useding to oral medicine, bee with height of the action look for thyme, the Greece and Rome artificial honey and plant thyme widely, the thyme also symbolizes brave, the style and grace, "the To smell of Thyme" praises language, at the ancient times Athens person, even soon afterward the ancient Rome person's, the society spreads most of view is bathing after at own of use thyme before the chest, the medicine grass learns a house toward melancholiac to recommend thyme, with heavy flap in low spirits spirit condition.

A musk still a lung enhances, "internal hour can smooth the stomach department to also remove flatulence".In addition, people's many years use it to come to the Qie phlegm and disinfect, and it still has the anti- fungi function.Can also take it while having a sore throat to make mouthwash, the result is fairly good!!

Plant characteristic:

The temperate zone region is familiar to go to the full mountain time wild thyme, the blossom of the amiability bloomed before early summer, landscape beautiful grand view.Be divided into erection and crawl two generally.In order to cover with a pale green color of the needle form, the leaf starts lignification and presents of the cent form from the Ji department.Follow the caulis minister to pay the thin pink lips form floret, the assortment also has lavender, red, white etc..There are numerous specieses, such as lemon thyme(the Lemon Thyme) and the silver spot thyme(the Silver Thyme)...etc..

Educate a method:

Sex pleased and alkaline soil, need sunlight ample, drain good.The seed is small, can use dissemination or insert an etc. method to breed, about about 10-15 days germination.Choose spring, the autumn sow seeds for two seasons, growing up slower the first year, the second year then grow up quickly and bloom.Can also adopt to transplant or the offshoot method breed, but among them"layering" result is the best, the livability is the most high, suggesting the raw recruit can use this method.

Adopt to accept conservancy:

Can pick at any time the caulis, use directly, or only use fresh leaf's slice.The caulis, leaf's aridity places to dry gradually in shade in the cool place nature, or whole pour to hang to dry by air, place in the bottle inside mark clearly date, and use early.

Extensive usage:

1.Cuisine:Leaf's slice can combine every kind of meat, the fish molluscs cuisine, matching the beef is also a delicacy;The biscuit pastry wait for every kind of food processing and all can make use of.

2.Health:Promote digest, recover physical strength, disinfect, antisepsis etc..

3.Hairdressing:Dissolve and can recover fatigue into the makeup water.

4.The tea drinks:The fresh vanilla or aridity all can use alone, or combine other vanilla plants to use together after.The fresh lemon thyme eating is special, the suggestion tries.

5.Ethereal oil:The leaf, flower, caulis can refine ethereal oil, there is result of fig uping the motion.

6.Flower arrangement:Can use in the dry flower and escort a flower and arrange flowers etc. various adornments up.

7.Horticulture:The ground top of the garage can plant to crawl a thyme, will have intention to surprisingly result.

8.Craft:Making use of or matching decorations with other vanillas alone all can express its special features.

【Body 】can promote circulatory system, reduce cholesterol and promote the leukocyte formation with strengthen resistibility, to chronic breath way disease, asthma, vein Dou Yan, larynx pain, catch a cold, develop a fever, have a headache, flow nose water, liver, digestive system, muscle ache, women's mastitis, suffer from insomnia the improvement of[with] etc. contain help.

【Mind 】 enhance nerve, activate brain cell and raise memory and attention.The cancellation is exhausted and resists a setback, stirs up courage and releases a mind.

【Hairdressing 】 the pimple, eczema, wound, the skin inflammation...etc. are all helpful.Scurf, skin of head Yang, repress to fall hair.

【Other 】 antisepsis characteristic.

1.     Take a bath slow stop and think with town through.

2.     Dissolve and can remove fatigue into the makeup water.

3.     Immerse and add honey and can cure cold and have a sore throat, stop coughing to turn the phlegm, protection breath way in, be suitable for processing tonsillitis, bronchitis particularly etc. condition of illness, can enhance lung;Can also stimulate the manufacturing of the leukocyte, help the body holdout disease, control a germ to spread, contribute to an immune system.

4.     Making tea can help to help digest and remove flatulence to also solve wine enough, having a help towards promoting and boosting digestive system particularly, can also improve the stomach department unwell cause of headache.

5.     The ability Jian turns skin of head, to scurf with repress to fall hair very much effectively

6.     Can make the wound, Chuang, eczema of the skin restore to original soon.

7.     Still have already disinfect, defended an insect and go to the function of the gamey smell, antisepsis besides.

8.     The fresh thyme tea fit melancholy and weariness drink, the morning replaces coffee by this vanilla tea particularly effectively, strain or work pressure big hour can also drink last.

9.     The skin quality of system excess secretes, keep to have no oil dumb light of the skin hydrate hydration and strengthen an activity

10.   Can promote digest, recover physical strength, protect the breath way, prevent catching a cold, stopping coughing to turn phlegm, ease women's menstrual cramp, promote and boost spirit, remove fatigue and enhance heart, anti- rheumatism.

But the pregnant woman should avoid drinking.

11.   Moisten, increase sheen

12.   Repress a collagen is resolved, reach the purpose of the pore delicacy.

13.   Repress a collagen is resolved, prevent pore from thin Wen creation thoroughly

14.   The strong power anti- oxygen, can insure the fatty acids composition is effectively absorbed by body

The musk grass Fen(thymol) is the main composition of the thyme ethereal oil, and the thyme ethereal oil be applied early is anti-virus in the antisepsis of use;The resveratrol(resveratrol, 3, 5, 4'-trihydroxystilbene) is to belong to polyphenol compounds, having an anti- to oxidize, prevent cancer and suppressing germ.This research aims at the musk grass Fen and resveratrol, through distinguish with the different density and the escherichia coliform(the Escherichia coli CCRC 10675 and E. The coli O157:the H7 CCRCs 15374)and the golden color staphylococcus(the Staphylococcus aureus CCRC 10780, CCRC 12655 and CCRC 13962) makes use of again the dilution method development to count after mixing the 1-40 mins.The result shows the musk grass Fen and resveratrol to each test germ stub it repress result, all manage the increment of the density and time everywhere and obvious exaltation.While handling to the 40 mins while increasing the musk grass Fen 125 ppms or the resveratrol 250 ppms alone, to E. The coli CCRC 10675 and E. The coli O157:the H7 CCRCs 15374 both have respectively 0.16, 0.78 logs and 0.26, the 0.18 log value disinfect power, mixing both to counteract to handle a 10-20 mins, then have 1.02~3.31 log value it to disinfect power, show the musk grass

BE repressed completely its growth namely when the mixture counteracts to handle a 1 min, can know the mixture counteracts a set it suppress the germ effect to surpass to increase the musk grass Fen or the resveratrol alone, show to have to add a function and strengthen it to suppress a germ activity.Apply after fresh food, soaking the bud of Mu Xiu and the mushroom slice through the ethanol aqua of the musk grass Fen or the resveratrol handling, in 4 ℃ store the period measurese to always get the germ variety of[with] few growths.Show as a result the musk grass Fen and resveratrol suppress the mushroom and the bud of Mu Xiu germ is live, all managing the increment of the density everywhere and raising obviously, with the result that 500 ppms handle best.Have while aiming at the bud of Mu Xiu and speech, handling a 30 mins by the musk grass Fen 500 ppms to also store to the 48 hs in 4 ℃ >1 log it suppress the germ effect, with the resveratrol of the same density handle the Mu Xiu bud have a 0.33 logs it to suppress the germ effect about;To mushroom, be with the musk grass Fen or the resveratrol 500 ppm processing and store a 48 hs in 4 ℃ , all have>3 logs it suppress the germ effect.In regard to same material, the musk grass Fen it suppresses the germ activity slightly big after the resveratrol of the same density, but this two compositions handling a 30 mins by the density with 125 ppms only, wasing stored by 4 ℃ 48 hs, mushroom and the bud of Mu Xiu of always get the germ number all low in or near with go to the control set handled by the ion water.Show the musk grass Fen in lower of the density 250 ppms, can reach certain degree it suppress germ an activity, and repress the microorganism survival of 4 ℃ store mushroom and the clover bud effectively, have further a potential of developing the application.

Be much more than only because the politician contain body odor, the musk grass has been the symbol of the courage from time immemorial, the plant scientific term"thymus" of the musk grass(thyme) comes from courage"thymon" of the Greek.

At the undemocratic society, the musk grass is the patent of the politician, in order to prevent the people ate courageous rebel.And in the democratic society, the politician only doesn't eat the musk grass every day, also taking a shower with the musk grass every day, because the musk grass can let the politician even stand to announce political views in vibrating a machine, the hand and foot can't shiver, either.In the meantime for the sake of Ying get ballot, the musk grass can make the politician have enough courage again thick and black of speak some such as the funny lies of"the edacity have to be all understood by fatty and swollen appearance to present, the but again says the oneself is like fairy self-containedly similar have never eaten";Open some such as the Arabian Nights of"free help everyone pack up can fly arbitrarily and freely of righteousness wings";Struggle do some such as the cartoon details of"attend and only can use the affair of honor of the knife sword, but polish off opponent with the big gun";Do some such as"in the septic tank swimming" doesn't behave to know of matter etc..

The musk grass the ancient time of above-mentioned increment courage spreads function, besides which, mutually the function of the Fu can also promote circulatory system, reduce cholesterol and promote the leukocyte formation to strengthen a resistibility.The musk grass mutually Fu to the chronic breath way disease, asthma, vein Dou Yan, larynx pain, catch a cold, develop a fever, have a headache, flow nose water, liver, digestive system, muscle ache, women's mastitis, suffer from insomnia the improvement of[with] etc. contain help.The category of the musk grass has a lot of also can use, have the most strong medicine grass quality by the wild musk grass of Englands among them.Mutually the Fu musk grass often steeps tea to be used for the help digest to reach agreement to take off hangover.Mutually the Fu musk grass tea also often adds honey to be used for the larynx pain, cold, the Ao breeze a cough...etc..The musk grass ethereal oil mutually the Fu can be used for the headache massage, can also make in to disinfect of spray.The musk grass and rosemary(rosemary) use together and outside mutually Fu and can clean scurf.

The musk grass' ising used especially is a sausage;The spice of the meat, fish etc. speciality.It also Be used for the Xun joss-stick(Raeucher) in ancient times.Its fragrance extract oil main musk of the ingredient grass Fen(Thymol), the musk grass Fen is anti-virus with resist the result for fermenting(Gaerungawidrig).Therefore formerly the musk grass use to digest a problem towards curing the stomach flatulence(Blaehung) and.The gram Nai river bank the pastor write a way:The musk grass ingredient is the ′′ very good stomach medicine ′′ , can also use to sweep chest.The county applies in the disease(the card his sex bronchitis Katarrhe(have a great deal of liquid with sticky Nong)), bronchitis(the Bron chitis), whooping cough(Keuch-husten) of the treatment a cough bronchitis(Husten-Bronchial) aspect in the musk grass.This plant's refining can cure halitosis(Desodoruerend).Then When the Black Death spread, the musk grass is used to prevent infection once and in great quantities.This according to its musk grass Fen, anti-virus, can be treated as the part is right.

Battlefield on the ancient Rome person front, will use what thyme steeps of water to bathe, hope to bring courage for them, so didn't despise it to grow small very short.It has rather sweet and strong medicine grass joss-stick, and thus of flavor, it is said the mind that can release durance, comfort the wound of people's mind.Also strengthen a resistibility, help the skin repair and maintenance, adjust reason the pimple skin quality, activate the brain cell, promote memory and attention, the anti- is upset and comfort mind wound.To scurf with repress to fall hair very much valid.

Thymol, a constituent of thyme essential oil, is a positive allosteric modulator of human GABAA receptors and a homo-oligomeric GABA receptor from Drosophila melanogaster

Abstract

1.                              The GABA-modulating and GABA-mimetic activities of the monoterpenoid thymol were explored on human GABAA and Drosophila melanogasterhomomeric RDLac GABA receptors expressed in Xenopus laevis oocytes, voltage-clamped at -60 mV. The site of action of thymol was also investigated.

2.                              Thymol, 1−100 M, resulted in a dose-dependent potentiation of the EC20 GABA response in oocytes injected with either 132s GABAA subunit cDNAs or the RDLac subunit RNA. At 100 M thymol, current amplitudes in response to GABA were 416 72 and 715 85% of controls, respectively. On both receptors, thymol, 100 M, elicited small currents in the absence of GABA.

3.                              The EC50 for GABA at 132s GABAA receptors was reduced by 50 M thymol from 15 3 to 4 1 M, and the Hill slope changed from 1.35 0.14 to 1.04 0.16; there was little effect on the maximum GABA response.

4.                              Thymol (1−100 M) potentiation of responses to EC20 GABA for 112s, 632s and 132s human GABAA receptors was almost identical, arguing against actions at benzodiazepine or loreclezole sites.

5.                              Neither flumazenil, 3-hydroxymethyl--carboline (3-HMC), nor 5-pregnane-3, 20-diol (5-pregnanediol) affected thymol potentiation of the GABA response at 132s receptors, providing evidence against actions at the benzodiazepine/-carboline or steroid sites. Thymol stimulated the agonist actions of pentobarbital and propofol on 132s receptors, consistent with a mode of action distinct from that of either compound. These data suggest that thymol potentiates GABAA receptors through a previously unidentified binding site.

Introduction

The rapid actions of the neurotransmitter GABA are mediated by ionotropic GABA receptors; these are pentameric transmembrane proteins with an integral, GABA-gated, anion channel. In vertebrates, all ionotropic GABA receptors are designated type A, and metabotropic receptors type B. Vertebrate GABAA receptors are confined to the nervous system, whereas insect ionotropic GABA receptors are present in the nervous system and on muscle cells. To date, 20 different vertebrate GABAA receptor subunit isoforms have been cloned: (1−6), (1−4), (1−3), (1), (1), (1), (1) and (1−3). Further diversity can arise due to alternative splicing of some subunit genes (for GABAA receptor classification see, for example,). The most common stoichiometry in mammalian brain is thought to be 221, although there is potential for considerable diversity of subunit composition. No data are available on the subunit stoichiometry of insect ionotropic GABA receptors, although three different subunit candidates are known to be expressed, one of which, RDL, has four splice variants. Subunit composition is an important determinant of the pharmacological and biophysical properties of recombinant GABAA receptors, and probably also of native insect GABA receptors.

The many known GABA receptor ligands include agonists, antagonists and modulators; positive allosteric modulators, for example, potentiate the actions of GABA. In humans and other mammals, behavioural effects which are typical of positive allosteric modulators of GABAA receptors include anxiolysis, cessation of convulsions, sedation and general anaesthesia, although some of these effects may result from simultaneous action at other receptor types. Some positive GABAA receptor modulators can also act as agonists on the same receptors when tested at higher concentrations and this activity may influence the spectrum of clinical effects observed. Most GABAA receptor potentiating compounds, with the notable exception of the benzodiazepine clonazepam, also enhance the action of GABA at native and recombinant insect GABA receptors, although they are often less potent than on GABAA receptors and lack the agonist activity observed at some vertebrate GABAA receptors. Insect GABA receptors are targets for several pesticides, such as dieldrin, lindane, BIDN and fipronil, all of which are antagonists. One insecticide analogue, -HCH, potentiates at GABA receptors, and this has been proposed to be acting via the barbiturate binding site on the receptor .

Thymol is a monocyclic phenolic compound, the usual natural source being the essential oil of Thymus vulgaris (Lamiaceae). Its main therapeutic application is in dental preparations to kill odour-producing bacteria. It is also employed as a preservative on the strength of its antimicrobialand antioxidant properties. Thymol has molluscicidal  and insecticidal properties. In the mollusc Lymnaea acuminata, lethal doses of thymol affected the activity of key nervous tissue enzymes, and this was postulated to be the cause of toxicity. As yet, no mechanism of action has been identified for thymol lethality, or that of related monoterpenoids, towards insects.

Recently, thymol was shown to have a direct agonist effect on heterologously expressed human GABAA receptors resembling that of the anaesthetic propofol. In this paper, we examine whether thymol, like propofol, also potentiates the activity of GABA at vertebrate GABAA receptors at lower concentrations than those required for agonist activity. We also test the actions of thymol at a recombinant insect GABA receptor, the Drosophila melanogaster RDLac subunit; this splice variant (one of four products of the Rdl gene) forms a functional homomeric GABA receptor and its pharmacology has been described in detail, including the actions of the insecticides dieldrin, lindane and fipronil. We also examine whether the site of action of thymol is shared by any other known GABA receptor modulators (benzodiazepines, barbiturates, pregnane steroids, loreclezole and propofol).

Methods

Investigations on insect and human GABA receptors were carried out in different laboratories, and this is reflected in minor differences in the respective protocols, as detailed in this section.

GABA receptor subunit cDNAs and cRNAs

Previous publications have described the cloning and sequencing of cDNAs encoding 1, 1, 3, 2 and 6 human GABAA receptor subunits, and also the Drosophila RDLac GABA receptor. Human cDNAs, encoding 1, 6, 3, 1 and 2s GABAA receptor subunits, were supplied by The Molecular Biology Department, Merck, Sharp and Dohme, Terling's Park, U.K. Wild-type Rdlac cDNA was a gift from Dr Richard Roush (Cornell University, U.S.A.); it had been inserted into the cloning vector pNB40. The plasmid was subcloned following established methods; subsequent extraction of pNB40 from E. coli was carried out using endotoxin-free, maxi-prep kits (Qiagen, U.K.). The plasmid was linearised with NotI restriction endonuclease to provide a transcription template, and RDLac cRNA was then synthesised with an SP6 RNA-polymerase and m7G(5')ppp(5')G capped using an 'mMessage mMachine' (Ambion), following the manufacturer's protocol.

Receptor expression in Xenopus oocytes

Human GABA receptor subunit combinations were expressed in Xenopus laevis oocytes. Mature female Xenopus oocytes (Blades, U.K.) were anaesthetised by immersion in a 0.4% solution of 3-aminobenzoic acid ethylester for 30−40 min, or until completely unresponsive, and part of the ovary was excised via a small abdominal incision. The isolated ovaries were immersed in modified Barth's solution (MBS) of the following composition (mM): NaCl, 88; KCl, 1; NaHCO3, 2.4; HEPES, 10; MgSO4 7H2O, 0.82; Ca(NO3)2 4H2O, 0.33; CaCl2 2H2O, 0.91; pH 7.5 (adjusted with NaOH), and then transferred to a hypertonic isolation medium composed of (mM): NaCl, 108; KCl, 2; EDTA, 1.2; HEPES, 10; pH 7.9 (adjusted with NaOH), to aid subsequent manual defolliculation. Residual follicular cells were removed by incubating the oocytes in collagenase type IA (Sigma, U.K.), 0.5 mg ml-1 in MBS, for 6 min. A manual oocyte injection pipette (Drummond, U.K.) was used to administer 20 nl of GABA subunit cDNA mixture to each cell nucleus. Combinations of three human GABAA receptor cDNAs were injected, in the ratios of either 1 : 1 : 1 or 1 : 0.1 : 1 to optimise the expression of benzodiazepine-sensitive GABAA receptors. The concentration of total cDNA in each case was 20 ng ml-1, in an injection buffer consisting of (mM): NaCl, 88; KCl, 1; HEPES, 15; pH 7 (adjusted with NaOH). Following injection, the cells were transferred to MBS supplemented with gentamycin, 50 mg l-1; penicillin, 10,000 U l-1; streptomycin, 10 mg l-1; and sodium pyruvate, 2.5 mM. Oocytes were maintained at 19°C initially.

Insect GABA receptors were expressed in Xenopus oocytes by a similar method. In this case, before injection, the ovaries were washed and stored in standard oocyte saline (SOS), of the following composition (mM): NaCl, 100; KCl, 2; CaCl2, 1.8; MgCl2, 1; HEPES, 5; pH 7.6 (adjusted with NaOH). The cRNA encoding RDLac, 50 ng at 1 gl-1, was injected cytoplasmically using a Nanoject pipette (Drummond, U.K.); some cells were omitted for use in control experiments or injected with the same volume of dH2O. The incubation medium employed consisted of SOS supplemented with antibiotics and pyruvate, at the concentrations described previously, and horse serum at 10 ml l-1. In the 30 min following injection, oocytes were kept at 4°C to allow recovery. Cells were incubated at 16°C and transferred to fresh medium on a daily basis.

Batches of cells responding with large currents were transferred to 4°C to prevent receptor overexpression and prolong viability.

Electrophysiology and data analysis

To investigate GABA receptor responses, oocytes were secured by a ring of stainless-steel entomological pins embedded in the Sylgard floor of a Perspex bath. Fresh bathing solution was continually perfused through the chamber by a gravity-fed system. All drugs were applied dissolved in the bathing solution, although stock solutions of hydrophobic compounds were prepared in DMSO or acetone and diluted in bathing solution such that the concentration of organic solvent did not exceed 0.1%. Solutions of 0.1% acetone or DMSO had no effect on the current required to clamp injected oocyte membranes at -60 mV, nor did they affect responses to GABA. DMSO and acetone, at these concentrations are used as solvents for drugs in oocyte electrophysiology.

Membrane currents recorded from oocytes expressing GABA receptors were measured by two-electrode voltage-clamp, with the membrane held at -60 mV, using 2 M KCl-filled electrodes with 1% agar in 2 M KCl at the tip for GABAA receptors, and 3 M KCL-filled electrodes for RDLac. Electrode resistance was maintained at 0.5−5 M . Currents due to GABAA receptors were amplified using a GeneClamp 500 Amplifier (Axon Instruments, U.S.A.) and recorded on two outputs: electronically, using 'Oocyte' for the Digitimer DigistoreTM System (Digitimer Ltd, U.K.), and on chart paper with a Thermal Arraycorder (WR 8500 series, Graphtec, U.K.). Currents through RDLac homomers were amplified using an Oocyte Clamp OC-725C amplifier (Warner Instrument Corporation, U.S.A.) and displayed on a chart recorder. Each cell expressing GABAA receptors was challenged with 3 mM GABA to obtain the maximal response, and those with a maximal response of less than 100 nA were rejected. This was not necessary for RDLac receptors, as consistently large responses were generated. Only oocytes yielding stable responses were selected for experimental work. Uninjected or distilled water-injected (dH2O-injected) oocytes did not respond to GABA or thymol.

Responses to drugs were measured at peak current. Dose−response data were generated using increasing concentrations of the ligand of interest. Curves were fitted to the data, both for individual cells and also to the mean data points. GraphPad Prism (GraphPad Software, U.K.) was used to fit the four-parameter logistic equation below, which describes a sigmoid curve of variable slope, to the normalised data:

where is the normalised current induced by a given concentration of agonist, [A]; Imax and Imin are the maximal and minimal normalised agonist responses, respectively; EC50 is the concentration of agonist predicted to elicit half the maximal response and nH is the slope (Hill) coefficient. Results are presented as the mean one standard error of the mean ( s.e.m.) of experiments on n cells. EC50 values given in the text for human GABAA receptors are mean values calculated from several EC50 values, each of which was estimated from the dose−response data obtained from an individual cell; for the insect RDLac receptor, the EC50 was estimated from dose−response data pooled from 14 cells. For the graphical presentation of data, all dose−response results were averaged before a single regression line was fitted. Differences between mean values were evaluated by unpaired or paired Student's t-test, or one-sample t-test, as appropriate, and considered significant if P<0.05.

Thymol dose−response experiments on human GABAA receptors were carried out by determining the GABA EC20 for each cell, and then applying the EC20 in conjunction with increasing doses of thymol after a 40 s preapplication with thymol alone. To estimate the GABA EC20 for RDLac homomers, dose−response data from 14 cells were pooled and used to fit a curve to the averaged data. For thymol dose−response experiments, a set concentration of thymol was applied for 2 min, followed by coapplication of this concentration with EC20 GABA. This regime was repeated using increasing concentrations of thymol. Data were handled in the same manner as for the GABA dose−response curves, except that each response was normalised to the EC20 GABA response for each cell.

Further investigations were carried out on human GABAA receptors. The effects of thymol on the GABA dose−response curve were estimated by applying increasing concentrations of GABA to each cell, and then applying the same GABA doses together with 50 M thymol, each after a 40 s thymol preapplication. To assess competitive interactions between thymol and GABA-inhibiting or -enhancing ligands, the GABA EC20 was applied in conjunction with the ligand in the presence and absence of 50 M thymol. To assess competitive interactions between thymol and the agonist-like effects of the positive modulators pentobarbital and propofol, the ligand was applied until stable responses were obtained, and then it was coapplied with 50 M thymol. In order to minimise desensitisation and rundown effects, washout periods between drug applications were 5 min after a maximal GABA response, and 3−10 min after other applications, depending on the drug and concentration applied.

Drugs

Propofol (2,6-diisopropylphenol, Aldrich), 3-HMC (Tocris), GABA (Sigma or Research Biochemicals Inc.), pentobarbital, 5-pregnanediol, thymol (Sigma), flumazenil (synthesised by K. Moore in the Medicinal Chemistry Department, Merck, Sharp and Dohme, Terling's Park, U.K.).

Results

Potentiation of GABA action by thymol on human GABAA receptors

Initial studies were performed on human 132s recombinant receptors as this subunit combination is abundant in the vertebrate CNS, and changing one or other of the subunits has a dramatic effect on the actions of modulators. Thymol, 1−100 M, applied prior to (for 40 s) and during the application of EC20 GABA resulted in dose-dependent potentiation of the GABA response. Above 100 M, thymol potentiation decreased with increasing thymol concentration. The maximal potentiation observed for the 132s GABAA receptor was 416 72% (n=5) at 100 M thymol.

Intrinsic activity of thymol on human GABAA receptors

Cells expressing 132s did not respond directly to 1−50 M thymol (the change in membrane current over the course of the 40 s application of thymol was never greater than 9 nA in amplitude). Thymol at 100 M and above generated responses, although these were extremely small in amplitude compared to the striking potentiation of the GABA-induced current by this same thymol concentration (as illustrated by the trace in. Dose−response curves showing the agonist action of thymol were not examined because the concentration of acetone required to solubilise 300 M and higher concentrations of thymol affected the current required to clamp the cell.

Thymol potentiates the GABA response mediated by recombinant, homomeric insect (Drosophila melanogaster) RDLac GABA receptors

Of all the splice variants of the Rdl gene, the Rdlac gene product has been most extensively studied. The GABA dose−response curve obtained from oocytes expressing RDLac was used to estimate an EC20 value for GABA of 4 M. This EC20 test concentration was then deployed in further experiments to investigate potentiation by thymol. The EC50 was 8.5 M (95% confidence interval: 7.1−10.0 M), which was close to previously published EC50 values for this receptor.

Thymol, 1−100 M, potentiated the EC20 GABA response when coapplied for 15−30 s, following a 2 min preapplication of thymol alone. The potentiation was fully reversible and dose-dependent. The degree of potentiation by thymol was also affected by the GABA concentration, thymol (100 M) increasing the response to 10 M GABA less than two-fold (data not shown), whereas the response to EC20 GABA (4 M) was potentiated over seven-fold.

Any possible potentiation by concentrations of thymol greater than 100 M could not be investigated due to instability of the clamped current at these doses. Thymol at 100 M was without effect when tested on uninjected oocytes (n=3, from three separate batches).

Thymol is a weak agonist on Drosophila melanogaster RDLac GABA receptors

A small-amplitude, direct agonist action of thymol was observed in oocytes expressing RDLac, but not in uninjected or dH2O-injected cells: Concentrations of 1−50 M thymol and below had a negligible effect, eliciting changes in the membrane potential of 15 nA or less over a 2 min application. Thymol, at 100 M, resulted in currents which were 57 22 nA (n=4) in amplitude at 2 min. Responses to thymol alone began immediately upon application, but were slow to gain amplitude and failed to plateau throughout the 2 min application time.

Effects of thymol on the GABA dose−response curve of human 132s GABAA receptors

Thymol potentiates the response to concentrations of GABA below EC100, the largest effects being seen over the range EC5−EC50. The amplitudes of currents induced by 3 and 100 M GABA were 7.6 1.7% (n=5) and 89.0 4.8% (n=5) of the maximum, respectively. In the presence of 50 M thymol, these responses were potentiated to 45.0 5.2% (n=5) and 98.6 3.0% (n=5) of the maximum GABA response, respectively. The GABA dose−response curve was shifted to the left by 50 M thymol and the mean EC50 for GABA was reduced significantly from 15 3 to 4 1 M (paired t-test). The Hill slope was also reduced significantly (paired t-test) from 1.35 0.14 to 1.04 0.16. However, 50 M thymol had little effect on the maximum GABA response.

Thymol potentiates the GABA responses mediated by human GABAA receptors of different subunit composition with similar potency

In the range 1−100 M, thymol potentiated the EC20 GABA response of 112s and 632s vertebrate GABA receptors in a dose-dependent manner. The thymol dose-response curves obtained for 112s, 632s and 132s receptors were almost identical: over the range of concentrations tested, GABA actions on the 3 subunit combinations were potentiated about equally by thymol.

Thymol-induced potentiation of 132s human GABAA receptors is not affected by the modulators flumazenil, 3-hydroxymethyl--carboline and 5-pregnanediol

Flumazenil is a competitive antagonist at the benzodiazepine site; flumazenil is not expected to depress the GABA response under normal circumstances, but instead reduces the potentiation of GABA responses by other benzodiazepines. Coapplication of thymol and GABA, following a 40 s preincubation with thymol, potentiated the GABA response to 346.5 34.7% (n=4). Application of 50 M thymol, EC20 GABA and 1 M flumazenil to oocytes expressing 132s receptors following preapplication of flumazenil (30 s) and thymol plus flumazenil (40 s) resulted in a mean increase of the EC20 GABA response to 276.3 6.6% (n=4). Mean values for thymol potentiation in the presence and absence of 1 M flumazenil were not significantly different (unpaired t-test), suggesting that thymol is not acting via the benzodiazepine site.

When applied to oocytes expressing 132s, the -carboline 3-HMC, 100 M, caused a slight decrease in the EC20 GABA response, as expected, the amplitude being 83.7 3% (n=3) of control. When challenged with 50 M thymol and EC20 GABA in combination, potentiation of the GABA response to 320 60% (n=3) of control values was observed. Coapplication of 50 M thymol, EC20 GABA and 100 M 3-HMC together, following preapplications of thymol and 3-HMC, resulted in an augmentation of the EC20 GABA response to 308 64% (n=3). The difference in the degree of thymol potentiation in the presence and absence of 3-HMC was not significant using an unpaired t-test, suggesting a lack of competition between thymol and -carbolines.

While there are no steroid site antagonists, the partial agonist 5-pregnanediol, at 3 M, applied with EC20 GABA, only increased the 132s GABA response to a small degree. The amplitude of the potentiated response was 164 26% (n=4) of control. The mean potentiation of the EC20 GABA response by 50 M thymol in these cells was 360 37% (n=4) of control. Coappli-cation of thymol, GABA and 5-pregnanediol, following preapplications of thymol and 5-pregnanediol (40 s), enhanced the control response to 395 39% (n=4). As the enhancement produced by thymol and 5-pregnanediol was not significantly lower than that of thymol alone (unpaired t-test), these data suggest that this steroid compound does not compete with thymol.

Actions of pentobarbital and propofol at 132s human GABAA receptors are enhanced by thymol

Pentobarbital and propofol potentiate GABAA receptors, and at saturating concentrations (300 and 60 M, respectively), both elicit inward currents in oocytes expressing the 132s receptor. Thymol, 50 M, applied for 40 s prior to coapplication with 300 M pentobarbital, significantly potentiated responses to pentobarbital (in a one-sample t-test) to 241 36% (n=5) when expressed as a percentage of the pentobarbital current. In a similar experiment, thymol, 50 M, was applied for 40 s prior to coapplication with 60 M propofol, a general anaesthetic with a similar structure to thymol. The response to propofol was also significantly enhanced in the presence of thymol (shown by one-sample t-test), but to a lesser degree than the pentobarbital response, showing 118 2.9% potentiation (n=4). These results suggest that thymol does not compete at the barbiturate- or propofol-binding sites.

Discussion

Thymol potentiates the actions of GABA at three recombinant human GABAA receptors of different subunit composition, and also at a recombinant insect ionotropic GABA receptor. Modulation of the human 132s, 632s and 112s GABAA receptors and DrosophilaRDLac homomers was dose-dependent over a similar concentration range, suggesting a non-subunit-selective action, although it appears that thymol is less potent on mammalian GABAA receptors than on the insect model ionotropic GABA receptor. At all subunit combinations, potentiation was observed at concentrations where thymol elicited either zero or minimal agonist activity, indicating that the enhanced response to GABA is likely to be the result of a positive allosteric action of thymol. A separate agonist action of thymol was seen at both mammalian and insect recombinant receptors between 100 and 500 M, and at these concentrations thymol failed to generate currents in uninjected or dH2O-injected eggs, indicating that its effects were GABA receptor-mediated. Agonist actions of thymol have also recently been described at rat 122 GABAA receptors expressed in HEK cells, although the potentiating effect was not investigated .

The significant leftward shift of the GABA dose−response curve obtained from human 132s-expressing oocytes demonstrates that thymol can decrease the apparent EC50 of GABA, but does not greatly affect the maximum response. This suggests that thymol is specifically facilitating the manner in which GABA binds to or activates the receptor, but cannot increase the current flow above the maximum possible achieved by GABA alone. Thymol also decreased the Hill coefficient (nH). In these respects, the actions of thymol on GABAA receptors are similar to that of the insecticide -HCH on RDLac homomers, but distinct from the action of -HCH on GABAA receptors. In the case of RDLac homomers, -HCH shifts the dose−response curve to the left, decreasing nH but without affecting the maximal response, whereas on GABAA receptors -HCH shifts the dose−response curve to the left and also depresses the amplitude of the maximal response.

Changing the receptor subunit composition of human GABAA receptors and observing the resulting impact on modulator potency is a rapid method of identifying specific subunits or subunit interfaces that are important for mediating the effects of a particular ligand. Such areas may form part of the modulator-binding site. Replacing 1 with 6 renders receptors otherwise sensitive to most benzodiazepines unresponsive to the majority of this class of compound, with the exception of certain benzodiazepine site ligands, such as the partial inverse agonist Ro 15-4513. The 1/6 substitution was used previously in an attempt to characterise a novel positive allosteric modulator of GABAA receptors, where the activity of (+)-ROD188 was compared on benzodiazepine-sensitive (rat 122s) and benzodiazepine-insensitive combinations (rat 12, rat 622) of subunits. In fact, (+)-ROD188 was most active at receptors containing the 6 isoform in x 22, where x=1, 2, 3, 5, 6. However, thymol showed no particular selectivity for 1 or 6 in terms of potency or maximal potentiation, suggesting that it does not act via the benzodiazepine-binding site, nor the (+)-ROD188 site. Loreclezole potentiates the actions of GABA at both RDLac and GABAA receptors, the determinants of potency at the loreclezole site on GABAA receptors being shared by the positive modulatory actions of etomidate and DMCM, but no competitive inhibitors have thus far been identified. Ligands binding to the loreclezole site are characterised by a reduced potency on 1-containing receptors in comparison to 3-containing receptors. Thymol potentiated the activity of GABA at recombinant 1 x 2s GABAA receptors, where x=1 or 3, with no difference in potency, suggesting that thymol does not interact in the same manner as loreclezole.

Studying competitive interactions between ligands is another method of classifying allosteric sites. Most benzodiazepines are positive allosteric modulators of vertebrate GABAA and native insect receptors but, with the exception of 4'-chlorodiazepam, are inactive or weakly active at RDLac homomers, probably because activity depends on the presence of more than one subunit type, as it does in vertebrates. It is relatively difficult to perform competition studies between two compounds that both potentiate GABA responses; therefore, the potent GABAA receptor benzodiazepine site antagonists flumazenil and the -carboline 3-HMC were chosen for study. Flumazenil (Ro15-1788), at 1 M, suppressed potentiation of the rat recombinant 122 GABAA receptor by the -carboline ZK 91085, but had no effect on the activity of (+)-ROD188, suggesting that ZK 91085 has a mode of action in common with benzodiazepines, but (+)-ROD188 does not. At invertebrate GABA receptors, 3-HMC was reported to competitively antagonise the potentiating effects of 4'-chlorodiazepam at RDL homomeric receptors. If thymol acted via the benzodiazepine -carboline-binding site, it might be expected that flumazenil or 3-HMC would displace it and result in a substantial reduction in thymol activity; no significant reduction in thymol activity occurred, suggesting that thymol does not act through either binding site.

Functional competition studies where both agents enhance the effects of the agonist are more difficult to interpret; it is not possible to predict exactly how the potentiating activities of two different compounds, whether acting at the same binding site or not, will be integrated by the receptor in terms of overall conformational changes and subsequent effect on the response. Previous studies have used the rationale that an interaction between compounds would manifest as a potentiation by the dual combination less than the (theoretical) additive effect of the two compounds applied separately. The partial steroid agonist 5-pregnanediol was used to investigate possible activity at the neurosteroid-binding site. The 5-pregnanediol-induced potentiation of the GABA response was much lower than that of thymol, as expected. As 5-pregnanediol is only weakly efficacious, competition between thymol and 5-pregnanediol could be expected to result in a combined enhancement lower than that produced by thymol alone; instead, the combined enhancement was higher. This result suggests that thymol does not act viathe steroid-binding site.

At GABAA receptors, barbiturates potentiate the effects of GABA and also have a direct agonist effect over a higher concentration range; since competition studies between two potentiating compounds yield complex results, we tested the ability of thymol to potentiate the agonist effects of pentobarbital and the anaesthetic propofol, ensuring saturation of the potentiating site. Like (+)-ROD188, thymol potentiated pentobarbital-induced currents, and, to a lesser degree, also those of propofol, indicating a lack of competitive interaction with these compounds. This was somewhat surprising since propofol and thymol are relatively similar in structure, and might be expected to share a site of action.

The evidence reported here suggests that thymol does not share sites of action with many of the most widely investigated allosteric modulators of GABAA activity, these being benzodiazepines, -carbolines, barbiturates, propofol, loreclezole and steroids. It is still contentious as to whether the barbiturates and propofol act as agonists and positive modulators via the same or distinct sites. A range of GABA receptor subunit point mutations influence both agonist and modulator activities of pentobarbitone and/or propofol, but this could reflect common transduction components rather than a single binding site . The simplest conclusion is that thymol has a mode of action different from that of pentobarbital and propofol as GABA receptor agonists, and possibly also as modulators.

When the effects of one drug are suppressed by another, in the absence of binding data, it is not possible to classify the interaction as competitive or allosteric inhibition. Furthermore, variations in drug potency between receptors composed of different subunit combinations may reflect differential transduction rather than differential binding. The lack of effect on the thymol enhancement in all of our experiments suggests that neither the binding site nor transduction domains mediating thymol potentiation were affected in any of the test conditions. In this study, thymol has tentatively been termed a positive allosteric modulator of GABA receptors even though no putative allosteric site has been defined. This term has previously been applied to ligands before a binding site on the receptor itself was confirmed, for example, (+)-ROD188 was described as a positive allosteric modulator of the GABA receptor, simply by the virtue that it stimulated GABA-induced currents at GABA receptors in a concentration-dependent fashion and induced negligible currents by itself (amplitude not given). No high-affinity binding sites were demonstrated for (+)-ROD188; a weak interaction with the benzodiazepine site was found, but was clearly not the site through which potentiation was mediated. Slight selectivity was shown for 6-containing receptors, but this does not necessarily reflect binding preferences. Until a specific binding site on the receptor is described, an intermediary site of action cannot be ruled out.

Intermediary sites of action have previously been proposed for other enhancers of GABA function, such as anaesthetics. Many chemicals that are volatile and lipophilic, physicochemical properties shared by thymol, have anaesthetic effects in humans; furthermore, at therapeutic concentrations, ion channels are the principal targets of anaesthetics, but for many years this was thought to be an indirect action secondary to bilayer disordering, for example, detergents and free fatty acids affect receptor channel function in this way. Thymol is known to affect plasma membrane properties such as stability and permeability to drugs, for example, piroxicam. In our study, thymol did not affect bilayer integrity at the concentrations required for potentiation, as negligible changes in the current required to clamp the membrane potential (<10 nA) occurred in uninjected oocytes.

The majority of evidence gathered recently supports anaesthetic action at discrete sites on the ligand-gated ion channels themselves. For example, although a huge diversity of structures enhance GABAA receptor function, within each chemical group there are strict structure−activity requirements and subunit preferences, and these observations are supported by competitive interactions between chemically related molecules in binding and functional studies. Thus far, it is not possible to draw any conclusions about the purported thymol-binding site on the GABA receptor. There could be multiple sites mediating the effect of thymol, for example, the portions of the GABA receptor protein mediating potentiation might be separate from those through which the agonist effect occurs, as proposed for other ligands that elicit both effects. As thymol does not appear to compete with other GABAergic ligands, it is possible that thymol enhancement occurs via a previously uncharacterised allosteric site on the GABAA receptor, which could represent a new avenue in therapeutic and/or pesticide research.