Pharmacology The Journal of Clinical

The Journal of Clinical Pharmacology http://jcp.sagepub.com/

Systemic availability and pharmacokinetics of thymol in humans C Kohlert, G Schindler, RW Marz, G Abel, B Brinkhaus, H Derendorf, EU Grafe and M Veit J Clin Pharmacol 2002 42: 731 The online version of this article can be found at: http://jcp.sagepub.com/content/42/7/731

Published by: http://www.sagepublications.com

On behalf of:

American College of Clinical Pharmacology

Additional services and information for The Journal of Clinical Pharmacology can be found at: Email Alerts: http://jcp.sagepub.com/cgi/alerts Subscriptions: http://jcp.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav

>> Version of Record - Jul 1, 2002 What is This?

Downloaded from jcp.sagepub.com at Humboldt -University zu Berlin on May 9, 2012

HERBAL MEDICINE

KOHLERTMEDICINE AVAILABILITY HERBAL ET AL AND PHARMACOKINETICS OF THYMOL

Systemic Availability and Pharmacokinetics of Thymol in Humans Claudia Kohlert, PhD, Gernot Schindler, MD, Reinhard W. März, PhD, Gudrun Abel, PhD, Benno Brinkhaus, MD, Hartmut Derendorf, PhD, FCP, Eva-Ulrike Gräfe, PhD, and Markus Veit, PhD

Essential oil compounds such as found in thyme extract are established for the therapy of chronic and acute bronchitis. Various pharmacodynamic activities for thyme extract and the essential thyme oil, respectively, have been demonstrated in vitro, but availability of these compounds in the respective target organs has not been proven. Thus, investigation of absorption, distribution, metabolism, and excretion are necessary to provide the link between in vitro effects and in vivo studies. To determine the systemic availability and the pharmacokinetics of thymol after oral application to humans, a clinical trial was carried out in 12 healthy volunteers. Each subject received a single dose of a Bronchipret® TP tablet, which is equivalent to 1.08 mg thymol. No thymol could be detected in plasma or urine. However, the metabolites

thymol sulfate and thymol glucuronide were found in urine and identified by LC-MS/MS. Plasma and urine samples were analyzed after enzymatic hydrolysis of the metabolites by headspace solid-phase microextraction prior to GC analysis and flame ionization detection. Thymol sulfate, but not thymol glucuronide, was detectable in plasma. Peak plasma concentrations were 93.1 ± 24.5 ng ml –1 and were reached after 2.0 ± 0.8 hours. The mean terminal elimination half-life was 10.2 hours. Thymol sulfate was detectable up to 41 hours after administration. Urinary excretion could be followed over 24 hours. The amount of both thymol sulfate and glucuronide excreted in 24-hour urine was 16.2% ± 4.5% of the dose. Journal of Clinical Pharmacology, 2002;42:731-737 ©2002 the American College of Clinical Pharmacology

E

Essential oils are mixtures of lipophilic, liquid, volatile, and often terpenoid compounds present in higher plants. More than 3000 compounds have been described so far.8 A broad variety of pharmacological activities of essential oils and their constituents have been investigated for potential use in medicine.9 One particular area of use for essential oils is respiratory medicine, where the oils of pine, eucalyptus, and thyme have a sound tradition and several clinical studies have provided evidence of efficacy.1-4,10-12 In particular, 1,8-cineol, originally a component of eucalyptus oil,10,12 and standardized myrtol2,4,11 have been subjected to pharmacological and clinical studies focusing on respiratory diseases. Thyme extracts, which are very popular in Germany for these disorders, have only a small record of clinical studies, but there is no dispute about their efficacy.1,3 Thyme extract products have shown clinical efficacy against acute bronchitis in comparison to synthetic compounds,3 as well as various activities in pharmacological assays. A dose-dependent anti-inflammatory effect of thyme extract was shown in carrageenin-induced

ssential oil compounds such as found in thyme extract are frequently used for the therapy of chronic and acute bronchitis. For these indications, several clinical trials have been carried out.1-4 Various pharmacodynamic activities for thyme extract and the essential thyme oil, respectively, have been demonstrated in vitro,5-7 but bioavailability of thyme oil ingredients has not been proven.

From the German Central Institute for Pharmaceutical Research (Dr. Kohlert, Dr. Gräfe, Dr. Veit), Sinzig, Germany; Department of Medicine I, Department of Complementary Medicine (Dr. Schindler, Dr. Brinkhaus), Friedrich-Alexander University, Erlangen-Nürnberg, Erlangen, Germany; Bionorica AG (Dr. Abel, Dr. März), Neumarkt/Opf., Germany; and College of Pharmacy (Dr. Derendorf), University of Florida, Gainesville. Supported by Bayern Innovativ, government funds, and Bionorica AG. Sites where the study was performed include the following: German Central Institute for Pharmaceutical Research; Friedrich-Alexander University, Erlangen-Nürnberg; and Bionorica AG. Dedicated to Professor Otto Sticher on the occasion of his 65th birthday. Submitted for publication September 28, 2001; revised version accepted March 3, 2002. Address for reprints: Dr. Markus Veit, German Central Institute for Pharmaceutical Research, Kranzweiherweg 10, 53489 Sinzig, Germany.

731

J Clin Pharmacol 2002;42:731-737


KOHLERT ET AL edema in rat paws.5 Antimicrobial activity against several gram-positive and gram-negative bacteria was shown for different thyme oils.7,13 By means of a bioimpedometric method, the bacteriostatic activity of thyme oil was determined.7 The essential oil of thyme—applied in concentrations from 50 ppm to 400 ppm—resulted in a reduction in the rate of growth of different bacteria. Concentrations of 200 ppm achieved more than 50% inactivation of the majority of microorganisms tested.7 Antibacterial activity against respiratory tract pathogens by gaseous contact was investigated recently.14 Petri dishes with thyme oil or thymol were placed in an airtight box, and inhibitory concentrations were measured. In addition, antiviral activities against influenza A virus and respiratory syncytial virus were found for thyme extract in the plaque reduction test.6 Recently, antioxidant effects of thyme oil and thymol in various rat tissues were reported.15 However, the clinical relevance of these activities depends on the systemic availability of these compounds in the respective target organs. Thus, investigation of absorption, distribution, metabolism, and excretion would provide a significant link between in vitro effects and in vivo efficacy. They may also be important in context of the safety of herbal medicinal products.16 Pharmacokinetics of most natural compounds such as thymol have not yet been investigated satisfactorily. For several monoterpenoid and phenylpropanoid compounds, there is a large amount of experimental data, but—especially with respect to humans— pharmacokinetic data are lacking.16 To evaluate whether thymol, the main compound of the essential oil of thyme, can contribute to the clinical efficacy observed for a preparation containing thyme extract (Bronchipret® TP tablets), a bioavailability study after oral administration of a single dose was carried out. This study should also provide preliminary data for further dose-finding studies and for the design of multiple-dose studies. SUBJECTS AND METHODS Subjects The study was carried out at the Department of Medicine I, Department of Complementary Medicine, Friedrich-Alexander University, Erlangen-Nürnberg (Erlangen, Germany). Twelve healthy male volunteers were recruited for the study after complete clinical examination. Routine blood and urine laboratory tests were performed. Mean age was 29.5 ± 6.74 years (mean ± SD), and mean body

mass index was 24.6 ± 2.0 kg m–2 (mean ± SD). Subjects were not allowed to use any medicine during the study. All subjects signed an informed consent form according to good clinical practice (GCP) guidelines. The protocol was approved by the ethics committee of the University of Erlangen, Germany. Study Design Each subject received a single dose of Bronchipret® TP tablets containing 60 mg of primrose dry extract (6.0-7.0:1; extracted by ethanol 47% (v/v)) and 160 mg of thyme dry extract (5.9-10.0:1, extracted by ethanol 50% (m/m)), which was batch-specific equivalent to 1.08 mg of thymol. The subjects were fasted at the time they received the medication. They stayed in the clinic for the first 15 hours of the study and returned to the clinic for regular blood sampling visits. Prestudies indicated that long sampling times would be necessary to cover the elimination phase completely. Therefore, a sampling period of 72 hours was chosen. During the entire time, they were on an essential oil-free diet, and they were not allowed to apply any cosmetics (e.g., toothpaste, aftershave, etc.) containing essential oil compounds to avoid interference with any other essential oil compounds. Therefore, the subjects were handed out a list with food and cosmetics they were not allowed to eat and apply during the time of the study period. Collection of Blood and Urine Samples Venous blood samples (9 ml per blood sample) were collected into EDTA tubes once before subjects were administered the medication and 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 24, 31, 38, 48, 55, 62, and 72 hours after administration. Blood was centrifuged for 10 minutes at 4000 × g. The supernatant plasma was transferred into reaction cups in aliquots of 0.5 ml, and 20 µl acetic acid 0.58 M were added to each aliquot for stabilization. The plasma was stored at –20°C until analysis. Urine was collected in plastic bottles in intervals of 0 to 3, 3 to 6, 6 to 9, 9 to 14, 14 to 24, 24 to 31, 38 to 48, 48 to 55, 55 to 62, and 62 to 72 hours. An aliquot of 50 ml of each sample was mixed with 0.1 g ascorbic acid as antioxidant and stored at –20°C until analysis. Analytical Methods For the determination of total concentration of thymol, thawed plasma samples were analyzed after enzymatic

732 • J Clin Pharmacol 2002;42:731-737 Downloaded from jcp.sagepub.com at Humboldt -University zu Berlin on May 9, 2012

AVAILABILITY AND PHARMACOKINETICS OF THYMOL

hydrolysis of conjugated thymol by headspace solid-phase microextraction (HS-SPME) prior to gas chromatographic analysis.17 For urine analysis, only 20 µl of acetic acid 0.58 M compared to 50 µl used in the plasma method were necessary to adjust to pH 5 because ascorbic acid was added before the samples were frozen. In addition, adaptation of the SPME extraction time to 25 minutes instead of 35 minutes for plasma analysis was performed, which is due to the different matrix. Validation of the methods was performed according to the FDA Draft Guidance for Industry No. 2578 (Bioanalytical Methods Validation for Human Studies). For both plasma and urine analysis, the international acceptance criteria were in the ranges required. At the limit of quantification (LOQ), within-day precision of the plasma and urine assay was below 19% and 12% (coefficient of variation [CV]), respectively, and below 5.6% (plasma) and 7.2% (urine) at higher concentrations. Accuracy was below 9% for plasma and below 13% for urine. Stability of both plasma and urine samples was given over 12 weeks at –20°C. The lower limit of quantification for plasma analysis was 8.1 ng ml–1 and for urine 10.9 ng ml–1. Plasma samples were analyzed in 12 runs and the urine samples in 5 runs. Each run was checked for linearity by a separate calibration curve. Precision and accuracy were controlled by running six external quality control samples (spiked blank plasma and urine samples with reference compounds) covering the whole range. The identification of phase II metabolites and the determination of free thymol in both plasma and urine were carried out exemplarily in 2 subjects each. For identification of the phase II metabolites, LC-MS/MS measurements were performed by A&M (Laboratory for Investigations in Analytics and Metabolism, Bergheim, Germany). Prior to LC-MS/MS (ionization mode: ESI negative), the following sample preparation procedures were applied. After addition of 550 µl acetonitrile to 550 µl plasma, the solution was vigorously shaken for 20 seconds and centrifuged at 7800 × g for 4 minutes. The solvent was removed under nitrogen stream at 40°C. The residue was dissolved in 5 µl acetonitrile and 45 µl 2 mM ammonium acetate by ultrasonication. After centrifugation at 7800 × g for 4 minutes, the supernatant was used for HPLC analysis. For urine sample preparation, 4 ml urine were acidified with 8 µl formic acid; 14 ml ethylacetate were added, and the samples were shaken for 2 minutes. For separation of the organic and aqueous phase, the samples were centrifuged at 7800 × g for 4 minutes, and the supernatant was evaporated under nitrogen stream at

40°C. The residues were dissolved in 12 µl acetonitrile and 108 µl 2 mm ammonium acetate by means of ultrasonication and used for HPLC analysis. Determination of free thymol in plasma was carried out by HPLC with a 12-channel coulometric array detector at the German Institute of Nutrition (BergholzRebrücke, Germany). To 400 µl of plasma (pH 5), 50 µl internal stock solution (o-Cresol, 4 µg ml–1), 50 µl water, and 500 µl methanol were added, vortexed, and centrifuged for 10 minutes at 7800 × g. The supernatant was used for HPLC analysis. Determination of free thymol in urine was carried out by GC-MS. n-Heptane (250 µl) was added to 500 µl of urine (pH 5) and vortexed for 20 minutes at 350 rpm. The supernatant was used for GC-MS analysis. Data Analysis Pharmacokinetic data were determined by noncompartmental analysis using Microsoft Excel based on the equations given by Gibaldi and Perrier.18 The maximum observed plasma concentration Cmax and the time to reach Cmax (tmax) were determined directly from the data. The AUC0 → clast was calculated using the linear trapezoidal rule. The apparent terminal elimination half-life was determined by linear regression of the terminal phase of the semilogarithmic plasma concentration versus time profiles. The mean absorption time (MAT) was calculated from 1/ka, which was obtained from ke and tmax by the Excel Solver function from tmax = ln(ka/ke)/(ka – ke). RESULTS Free thymol could not be detected in human plasma. By means of LC-MS/MS analysis, only thymol sulfate but not the glucuronide was identified in human plasma (reference compound proved feasibility) (Figure 1). The plasma concentration versus time curves of thymol sulfate (measured by LC-MS) and total thymol concentrations after enzymatic hydrolysis of plasma (measured by HS-SPME) showed parallel profiles (Figure 2). Hence, the quantification of thymol in plasma was based on the total thymol concentration after enzymatic hydrolysis of the sulfate. No free thymol was found in urine either. Instead, two phase II conjugates were identified by LC-MS/MS as thymol sulfate and thymol glucuronide (Figure 1). The ratio of peak areas of thymol sulfate and thymol glucuronide was constant over the different urine fractions (Figure 3). The time course of the total thymol concentrations in human plasma is shown in Figure 4. Pharma733

HERBAL MEDICINE


KOHLERT ET AL

peak area

thymol sulfate thymol glucuronide

RO

Compound

R

3

9

6

14

time [h]

-------------------------------- -----------------------------thymol

Figure 3. Cumulative renal excretion of thymol sulfate and thymol glucuronide in 1 subject.

H—

thymol sulfate

HO3S thymol glucuronide

HOOC HO HO

O OH

Figure 1. Structures of thymol, thymol sulfate, and thymol glucuronide.

plasma concentration versus time profile was biphasic, subdivided into a distribution phase and a slow terminal elimination phase beginning at about 10 hours after administration and lasting up to an average of 38 hours. Elimination half-life was calculated to be 10.2 ± 1.4 hours (mean ± SD) (Table I). In urine, the elimination of thymol conjugates was detectable for the first 24-hour interval, with most being eliminated after 6 hours. The combined amount of both thymol sulfate and glucuronide excreted in 24-hour urine was 16.2% ± 4.5% of intake. The renal clearance was calculated to be 0.271 ± 0.7 L h–1.

Cp [ng.mL-1]

thymol sulfate [response/250000]

DISCUSSION 140 120 100 80 60 40 20 0 0

1

2

3

4

5

6

t [h]

Figure 2. Thymol plasma concentration determined after enzymatic hydrolysis of plasma ( ) and thymol sulfate measured by LC-MS in 1 subject ( ).

cokinetic data analysis was performed with only 11 subjects, as thymol was already detected in the blank samples (plasma and urine) of 1 subject. Thymol was rapidly absorbed. Thymol sulfate could be detected in plasma 20 minutes after application. Maximum plasma levels of 93.1 ± 24.5 ng ml–1 (mean ± SD) were reached after 1.97 ± 0.77 hours (mean ± SD) (Table I). The

Thymol was present in human plasma only as thymol sulfate. Hence, the quantification of thymol was based on the thymol concentration obtained after enzymatic hydrolysis. Intake of one Bronchipret® TP tablet resulted in plasma concentrations that showed the same profile among all subjects. Thymol was absorbed quickly. Considerable plasma concentrations of thymol sulfate could already be detected after 20 minutes. This fast absorption indicates that thymol is mainly absorbed in the upper part of the gut. This confirms previous observations made by Somerville et al19 with ileostomy patients. In this study, the renally excreted amount of menthol after oral intake of peppermint oil by ileostomy patients was only 11% less compared to healthy volunteers. After oral application of 1,8-cineol and α-pinene, these compounds were detectable after 10 to 30 minutes in plasma as well.20,21 This fast absorption of essential oil compounds might have been favored by the small size and the lipophilic characteristics of the molecules. Maximum plasma concentrations of 93.1 ng ml–1 were reached after 1.97 hours (Table I), which is in accordance



Table I Pharmacokinetic Data of Total Thymol Absorption and Elimination in Human Plasma after Single Oral Administration of One Bronchipret® TP Tablet Mean

Dose (mg) Cmax (ng/ml) tmax (h) t1/2 (h) AUC0 → clast (ng h/ml) MRTabs (h) MAT (h) CLtot/f (L/h) Vdss/f (L) Vdarea/f (L)

SD

Minimum Value

1.08 93.11 24.47 1.97 0.77 10.2 1.4 837.3 278.5 12.6 2.1 0.53 0.04 1.2 0.3 14.7 5.1 17.7 5.6

55.90 0.80 8.3 456.7 8.1 0.46 0.8 6.1 10.8

Median

Maximum Value

Geometric Mean

Number

99.01 2.03 9.9 835.8 12.5 0.54 1.1 13.8 17.2

125.82 3.13 12.9 1281.6 15.2 0.59 1.8 23.3 29.5

90.04 1.81 10.1 793.6 12.4 0.53 1.2 13.9 16.9

11 11 11 11 11 11 11 11 11

Cmax, peak plasma concentration; tmax, time to reach Cmax; t1/2, elimination half-life; AUC0 → clast, area under the concentration-time curve from time 0 to clast; MRTabs, mean residence time after extravascular administration; MAT, mean absorption time; CLtot/f, total body clearance with respect to unknown bioavailability f; Vdss/f, volume of distribution at steady state with respect to unknown bioavailability f; Vdarea/f, volume of distribution during the elimination phase with respect to unknown bioavailability f.

1000 mean Cp [ng .mL -1]

median

100

10

1 0

5

10

15

20

25

30

35

30

35

40

tim e [h]

120 mean median

Cp [ng.mL-1]

100 80 60 40 20 0 0

5

10

15

20

25

40

time [h]

Figure 4. Thymol concentration (after enzymatic cleavage) in plasma (mean ± SD; median) of 11 volunteers after administration of ® one Bronchipret TP tablet.

with the tmax from other essential oil compounds after oral intake.20,21 Thymol plasma concentrations— after enzymatic hydrolysis of thymol sulfate—showed a biphasic profile like most of the essential oil com-

pounds investigated.22-25 The terminal elimination phase set in after 10 to 12 hours, and thymol could be detected up to an average of 38 hours. Elimination half-life was determined to be 10.2 hours (Table I). Other compounds such as 1,8-cineol and α-pinene showed elimination half-lives of 3.6 and 5.8 hours, respectively.20,21 Although plasma levels were detectable up to an average of 38 hours, the renal elimination of thymol conjugates was completed within 24 hours. This discrepancy might be due to the fact that very small amounts of renally eliminated thymol conjugates could not be quantified any more after 24 hours (< LOQ). The fact that thymol sulfate is eliminated slowly can also be deduced from the small clearance (Cltot/f) of 1.2 L h–1. The renal clearance of 0.271 L h–1 indicates high protein binding and/or reabsorption in the kidney, respectively. The volume of distribution (Vdss/f) of 14.7 L indicates that thymol sulfate stays mainly in the extracellular space (Table I). The bioavailability of thymol sulfate after administration of thymol is at least 16%, because 16% of the dose administered was excreted into the urine as thymol conjugates. However, since Vdss/f is only 14.7 L, a much larger number for f is likely. Thymol was present in human plasma only in its conjugated form. Our LC-MS/MS experiments gave evidence only for thymol sulfate; the glucuronide was not detected. However, for other phenolic compounds, there is evidence for both metabolites. After oral application of a mixture of phenol, guaiacol, p-cresol, and creosol (15-32 mg each), formation of glucuronide and—except for creosol—sulfate was observed in serum.26 The absence of thymol glucuronide in plasma 735

HERBAL MEDICINE


KOHLERT ET AL

could be the result of lower activity of hepatic UDPglucuronyltransferase compared to sulfotransferase. Thus, conjugation to sulfuric acid and glucuronic acid would compete, and only after application of much higher doses, formation of glucuronide could be observed.26 Free thymol was not present in human plasma, which is in accordance with investigations from Ogata et al.26 Despite much higher doses of phenols, only a small amount of free phenols could be detected in serum. The data available so far indicate that thymol is systemically available only as thymol sulfate. In urine, both thymol glucuronide and thymol sulfate were present, which confirms previous observations made by Takada et al.27 After oral application of thymol to humans and rats, both metabolites were identified in urine, but confirmation of structure by MS was lacking. Phenol applied intravenously to mice resulted in excretion of both metabolites, with sulfate being the main metabolite. Increasing dose shifted the ratio from sulfate toward glucuronide.28 For phenol, guaiacol, p-cresol, and creosol, formation of both conjugates—except for creosol—was observed.26 Free thymol was not found in urine. After oral application of 500 mg menthol to volunteers, no free menthol could be detected in urine.29 Unconjugated phenol, guaiacol, p-cresol, and creosol were found in urine only in small amounts, although the dose was more than 15 times higher than applied in our study.26 Data available so far indicate that—despite its absence in plasma—thymol glucuronide is eliminated renally. For other compounds (e.g., probenecid), the presence of the glucuronide in urine was proven, although it was not detected in plasma.30,31 In the case of thymol, thymol sulfate could be reabsorbed in the proximal tubule after glomerulary filtration. Cleavage to thymol could be achieved by the activity of arylsulfatases, which was proven for human kidney tissue.32 On the other hand, it is conceivable that the cleavage to thymol is achieved by enzymes located at the luminal brush border.33,34 Thus, thymol could be subsequently reabsorbed. Glucuronidation of thymol could be performed by UDP-glucuronyltransferases followed by secretion into the proximal tubule. Investigations in human kidney microsomes point to this mechanism. Although the liver is considered the most important organ for biotransformation, recently, kidney microsomes have demonstrated more effective glucuronidation of phenolic compounds than liver microsomes or intestinal microsomes.35,36 Extrahepatic glucuronidation was also proven for morphine37 and for propofol,35 which is structurally similar to thymol. In addition, renal

glucuronidation of paracetamol,34 1-naphtol,38 and 4-dimethylaminophenol39 was demonstrated by means of the isolated perfused rat kidney. Furthermore, variable activities of UDP-glucuronyltransferases were demonstrated for different tissues.36 The constant ratio of sulfate and glucuronide over the different urine fractions supports the hypothesis of renal cleavage of thymol sulfate with subsequent conjugation to glucuronic acid. However, to confirm this hypothesis, further investigations concerning the mechanism of renal metabolism are necessary. The data available so far indicate that a pharmacological effect observed in vivo after oral administration of a preparation containing thyme extract would be due to thymol sulfate. However, the pharmacodynamic effects of this conjugate have not been investigated yet. The pharmacodynamic effect is not necessarily exerted by conjugates present in plasma as they may be cleaved at the site of action by sulfatases. Activity of this group of enzymes has been proven in human lung tissue for a number of model compounds.40 Thus, pulmonary elimination of thymol despite the absence of free thymol in human plasma could be explained.41 This way, free thymol could be effective at the target organ respiratory system. Evidence of cleavage of sulfate to thymol would provide a further link between in vitro and in vivo results. In addition, the pharmacodynamic activity of potential phase I metabolites of thymol as observed in rats42 would be of great interest for understanding its clinical efficacy. Dedicated to Professor Otto Sticher on the occasion of his 65th birthday.

REFERENCES 1. Knols G, Stal PC, Van Ree JW: Productive coughing complaints: Sirupus Thymi or Bromhexine? A double blind randomized study. Huisarts Vet 1994;37:392-394. 2. Meister R, Zimmermann T: Einfluß einer 6 monatigen Therapie mit Myrtol standardisiert auf den Krankheitsverlauf der chronischen Bronchitis. Ergebnisse einer multizentrischen placebokontrollierten Doppelblindstudie. Pneumologie 1996;50:90. 3. Ernst E, März R, Sieder C: A controlled multi-centre study of herbal versus synthetic secretolytic drugs for acute bronchitis. Phytomedicine 1997;4:287-293. 4. Matthys H, de Mey C, Carls C, Rys A, Geib A, Wittig T: Efficacy and tolerability of myrtol standardized in acute bronchitis: a multi-centre, randomised, double-blind, placebo-controlled parallel group clinical trial vs. cefuroxime and ambroxol. Arzneimittel Forsch 2000;50:700-711. 5. Morgenstern E: Die antiphlogistische Wirkung von Bronchipret und seiner Komponenten im Rattenpfoten-Ödem-Modell, in: März R (ed.), Bronchitis—Neue Erkenntnisse zu Wirkungen und Wirksamkeit von Arzneipflanzen. Freiburg: Karger, 1998.



6. Schwenk U: Antivirale Wirkung von Bronchipret-Tropfen und Thymianextrakt, in: März R (ed.), Bronchitis—Neue Erkenntnisse zu Wirkungen und Wirksamkeit von Arzneipflanzen. Freiburg: Karger, 1998. 7. Marino M, Bersani C, Comi G: Antimicrobial activity of the essential oils of Thymus vulgaris L. measured using a bioimpedometric method. J Food Protect 1999;62:1017-1023. 8. Teuscher E: Biogene Arzneimittel. Stuttgart: Wissenschaftliche Verlagsgesellschaft, 1997. 9. Schilcher H: Effects and side-effects of essential oils, in: Svendsen AB, Scheffer JJ (eds.), Essential Oils and Aromatic Plants. Dordrecht, the Netherlands: Nijhoff, 1985. 10. Grimm H: Antiobstruktive Wirksamkeit von Cineol bei Atemwegserkrankungen. Therapiewoche 1987;37:4306-4311. 11. Federspil P, Wulkow R, Zimmermann T: Wirkung von Myrtol standardisiert bei der Therapie der akuten Sinusitis—Ergebnisse einer doppelblinden, randomisierten Multicenterstudie gegen Placebo. Laryngo Rhino Otol 1997;76:23-27. 12. Dorow P: Chronisch-obstruktive Bronchitis. Welchen Einfluß hat Cineol auf die mukoziliäre Clearance? Therapiewoche 1989;39: 2652-2654. 13. Pattnaik S, Subramanyam VR, Kole C: Antibacterial and antifungal activity of ten essential oils in vitro. Microbios 1996; 86:237-246. 14. Inouye S, Takizawa T, Yamaguchi H: Antibacterial activity of essential oils and their major constituents against respiratory tract pathogens by gaseous contact. J Antimicrob Chemoth 2001;47: 565-573. 15. Youdim K, Deans S: Dietary supplementation of thyme (Thymus vulgaris L.) essential oil during the lifetime of the rat: its effects on the antioxidant status in liver, kidney and heart tissues. Mech Ageing Dev 1999;109:163-175. 16. Kohlert C, van Rensen I, März R, Schindler G, Graefe EU, Veit M: Bioavailability and pharmacokinetics of natural volatile terpenes in animals and humans. Planta Med 2000;66:495-505. 17. Kohlert C, Abel G, Schmid E, Veit M: Determination of thymol in human plasma by automated headspace solid-phase microextraction/ gas chromatographic analysis. J Chromatogr B 2001;7:11-18. 18. Gibaldi M, Perrier D (eds.): Pharmacokinetics. New York: Marcel Dekker, 1982. 19. Somerville KW, Richmond CR, Bell GD: Delayed release peppermint oil capsules (Colpermin) for the spastic colon syndrome: a pharmacokinetic study. Brit J Clin Pharmacol 1984;18:638-640. 20. Zimmermann T, Seiberling M, Thomann P, Karabelnik D: Untersuchungen zur relativen Bioverfügbarkeit und zur Pharmakokinetik von Myrtol standardisiert. Arznemittel Forsch 1995;45: 1198-1201. 21. Langeneckert A: Untersuchungen zur Pharmakokinetik und relativen Bioverfügbarkeit von alpha-Pinen, 1,8-Cineol und Menthol nach dermaler, inhalativer und peroraler Applikation ätherischer Öle. PhD dissertation, Johann Wolfgang Goethe-Universität, Frankfurt am Main, 1998. 22. Kleinschmidt J, Römmelt H, Zuber A: The pharmacokinetics of the bronchosecretolytic ozothin after intravenous injection. Int J Clin Pharmacol Ther Tox 1985;23:200-203. 23. Schuster O, Haag F, Priester H: Transdermale Absorption von Terpenen aus den etherischen Ölen der Pinimenthol-S-Salbe. Med Welt 1986;37:100-102.

24. Falk A, Hagberg M: Uptake, distribution and elimination of alphapinene in man after exposure by inhalation. Scand J Wor, Env Hea 1990;16:372-378. 25. Jager W, Nasel B, Nasel C, Binder R, Stimpfl T, Vycudilik W, et al: Pharmacokinetic studies of the fragrance compound 1,8-cineole in humans during inhalation. Chem Senses 1996;21:477-480. 26. Ogata N, Matsushima N, Shibata T: Pharmacokinetics of wood creosote: glucuronic acid and sulfate conjugation of phenolic compounds. Pharmacology 1995;51:195-204. 27. Takada M, Agata I, Sakamoto M, Yagi N, Hayashi N: On the metabolic detoxification of thymol in rabbit and man. J Toxocol Sci 1979; 4:341-350. 28. Kenyon EM, Seeley ME, Janszen D, Medinsky MA: Dose-, route-, and sex-dependent urinary excretion of phenol metabolites in B6C3F1 mice. J Toxicol Env Health 1995;44:219-233. 29. Bell G, Henry D, Richmond C: A specific g.l.c. assay for menthol in urine. Brit J Clin Pharmacol 1981;12:281. 30. Vree TB, Hekster YA, Anderson PG: Contribution of the human kidney to the metabolic clearance of drugs. Ann Pharmacother 1992; 26:1421-1428. 31. Vree TB, van Ewijk-Beneken Kolmer EW, Wuis EW, Hekster YA, Broekman M: Interindividual variation in the capacity-limited renal glucuronidation of probenecid by humans. Pharm World Sci 1993; 15:197-202. 32. Munroe DG, Chang PL: Tissue-specific expression of human arysulfatase-C isozymes and steroid sulfatase. Am J Hum Gen 1987; 40:102-114. 33. Cavallini M, Palestini M, Antonucci A, Proia G, Tersigni R: Urinary excretion of arylsulfatases A and B in patients with renal transplants. Minerva Chir 1980;35:727-730. 34. Hart S, Calder I, Ross B, Tange J: Renal metabolism of paracetamol: studies in the isolated perfused rat kidney. Clin Sci 1980;58:379-384. 35. Raoof AA, van Obbergh LJ, de Ville de Goyet J: Extrahepatic glucuronidation of propofol in man: possible contribution of gut wall and kidney. Eur J Clin Pharmacol 1996;50:91-96. 36. Shipkova M, Strassburg CP, Braun F, Streit F, Gröne HJ, Armstrong VW, et al: Glucuronide and glucoside conjugation of mycophenolic acid by human liver, kidney and intestinal microsomes. Brit J Clin Pharmacol 2001;132:1027-1034. 37. Mazoit JX, Sandouk P, Scherrmann JM, Roche A: Extrahepatic metabolism of morphine occurs in humans. Clin Pharmacol Ther 1990;48:613-618. 38. Redegeld FA, Hofmann GA, Noordhoek J: Conjugative clearance of 1-naphtol and disposition of its glucuronide and sulfate conjugates in the isolated perfused rat kidney. J Pharmacol Exp Ther 1988;244:263-267. 39. Elbers R, Kampffmeyer HG, Rabes H: Effects and metabolic pathway of 4-dimethylaminophenol during kidney perfusion. Xenobiotica 1980;10:621-632. 40. Toussaint C, Albin N, Massaad L, Grunewald D, Parise O, Morizet J, et al: Main drug- and carcinogen-metabolizing enzyme systems in human non-small cell lung cancer and peritumoral tissues. Cancer Res 1993;53:4608-4612. 41. Bischoff R: Non-invasive exhalation monitoring—analysis of volatile compounds at the ppb and sub-ppb level. Phytomedicine 2000; 7(Suppl. 2):29. 42. Austgulen L, Solheim E, Scheline R: Metabolism in rats of p-cymene derivatives: carvacrol and thymol. Pharmacol Toxicol 1987;61:98-102.

737

HERBAL MEDICINE
