Spasmolytic effects of Thyme

2015

The spasmolytic properties are commonly considered as the major action of thyme preparations. In this regard Thymus vulgaris is the most representative species. Therefore many publications have focused on the effects of thyme preparations on smooth muscles, especially rat and guinea pig intestines, such as duodenum and ileum, guinea pig trachea.. seminal vesicles and rabbit jejunum. Two different protocols are typically followed: (i) The isolated smooth muscle is first contracted using several agonists (acetylcholine, histamine, adrenaline, nicotine and BaCl2) and the thyme preparations are subsequently added until maximum relaxation is achieved. The spasmolytical effect is evaluated by measuring the maximum relaxant effect and the ED50 (contraction that produces 50 per cent of the maximum spasmolytic response), (ii) The isolated smooth muscle is first incubated with the thyme preparations; the modification of the dose-response curves produced by the contracting agents are calculated. In this protocol, the relaxant agent remains in the bath throughout the experiment. The use of various spasmogens with different mechanisms of action causing muscle contraction can provide information on the pharmacological basis of the spasmolytic properties. As reference substances atropine, papaverine, and isoprenaline are used.

Spasmolytic activity of the essential oil of thyme (Thymus vulgaris)

Debelmas and Rochat have shown that thyme oils in which phenols are the predominant components have antispasmodic activity on intestinal smooth muscle contracted by several agents. Later the same authors studied the spasmolytic activity of the oils of different plants using various isolated smooth muscles and contractor agents. They found that thyme oil was the most active, presenting an antispasmodic action of an unspecific type inhibiting the contractions induced by all agents tested. These initial studies were carried out with water saturated with the essential oil, trying to overcome the problems to bring the hydrophobic essential oil into contact with the isolated organ.

Reiter and Brandt studied the effects of the volatile oils of 22 plants from 11 different families on tracheal and ileal smooth muscles of the guinea pig. All the oils showed relaxant effects on the tracheal smooth muscle developing shortly after addition. The most potent oils were (in the order of potency): angelica root, clove, elecampane, basil and balm. Sixteen oils inhibited the phasic contraction of ileal preparations with ED50 values between 4.5 and 76mg/l. With regard to the relaxant effects, the majority of oils were 1.4—8.4 times more potent on the ileal than on the tracheal muscle. This ratio was almost eight-fold for the essential oil of Thymus vulgaris. The authors divided the 22 volatile oils investigated into three groups according to their different effects on the mechanical behaviour of the stimulated ileal mysenteric plexus-longitudinal muscle preparations. Together with 15 others thyme oil belongs to the first group which had predominantly relaxing effects.

Spasmolytic activity of the essential oils of different Thymus species

Between 1985 and 1990 our research group carried out several studies aimed at studying spasmolytic activities of a variety of essential thyme oils from plants collected in eastern Andalusia, relating them with their components. In Table “Relaxant effects (ED50) versus contractions induced by acetyl-choline in isolated rat duodenum and quantitative composition of different essential oils of thyme when collected in full bloom”, the ED50 values of these essential oils and the chemical compositions (only functional groups) are given. All the oils produced a relaxant effect against acetylcholine-induced contractions in isolated rat duodenum. However marked differences existed which might partly be explained by the differences of the chemical composition. One may highlight the following:

  • Oils containing higher portions of phenolic components have shown higher spasmolytic potency, namely the oils of Thymus zygis () and Thymus orospedanus.
  • The oil of Thymus granatensis although lacking phenolic compounds showed a powerful relaxant effect. This could be due to the high content of terpene hydrocarbons, which also may explain the high effect of Thymus orospedanus containing both, phenols and hydrocarbons.
  • Essential oils lacking phenolic components and with low hydrocarbon levels are less potent, documented by the results obtained with oils of Thymus baeticus () and Thymus longiflorus ().
  • The lower spasmolytic effect of Thymus longiflorus () in comparison with that of Thymus baeticus () may be due to a higher level of ethers (1,8-cineole).

The above results prompted our group to investigate the spasmolytic potency in more detail (Table Relaxant effects (ED50) of diffetent components of essential oils versus contractions induced by acetylcholine and BaCl2 in isolated rat duodenum. The relative potency was calculated in comparison to papaverine). Taking all the data obtained into consideration one can say that indeed the phenolic components (thymol and carvacrol) as well as the terpene hydrocarbons (myrcene and caryophyllene) presented higher spasmolytic potency. Camphor was shown to be inactive whereas 1,8-cineole acted as a spasmogenic and showed to increase rat duodenum contractions up to a maximum of 75 per cent of acetylcholine induced contractions. This effect was competitively antagonised by atropine. Consequently these results favour the hypothesis that 1,8-cineole acts as a partial agonist at the level of the acetylcholine receptors and that the presence of 1,8-cineole in the oils diminishes their spasmolytic capacity.

Table Relaxant effects (ED50) of diffetent components of essential oils versus contractions induced by acetylcholine and BaCl2 in isolated rat duodenum. The relative potency was calculated in comparison to papaverine

Agonist Antagonist ED50 (µM) Relative potency
Acetylcholine Carvacrol 6.67 + 0.86 1.28
Thymol 4.88±0.74 1.75
Myrcene 5.48 ±1.08 1.56
Caryophyllene 9.00±2.14 0.95
Camphor inactive
Papaverine 8.55 + 1.09 1
BaCl2 Carvacrol 7.73±1.22 0.41
Thymol 7.25±0.81 0.44
Myrcene inactive
Caryophyllene 1.34 + 0.25 2.37
Camphor inactive
Papaverine 3.18 + 0.68 1

Note: The components of volatile oils were emulsificied in water by means of tween 20 at a proportion of 9/1 p/p.

The mechanism of the spasmolytic effect was studied by the modification of the dose-response curves induced by acetylcholine and CaCl2. All the essential oils included in Table modified the dose-response curves induced by acetylcholine, showing a dosage-dependent decrease in the maximum effect suggesting a non-competitive mechanism. Such oils are capable of inhibiting the contractions induced by CaCl2 in high-KCl Ca -free solution, also diminishing their maximum effect in a dose-dependent way. Godfraind demonstrated that Ca2+-induced contractions of a KCl-depolarized smooth muscle are due to an increased Ca influx through voltage-stimulated type-L Ca2+ channels. Therefore, the inhibitory effects of essential oils in these concentrations may also be explained by a) an inhibition of Ca entry through voltage-stimulated channels into the smooth muscle and/or b) blocking release of intracellular bound Ca +.

Several authors have investigated the spasmolytic mechanism of some phenolic compounds of essential oils. Van den Broucke and Lemli and Cabo studied the antagonistic effect of thymol and carvacrol on guinea pig ileum and rat duodenum against the contractions induced by carbachol, histamine and BaCl2 and concluded that contractions induced by these spasmogenic agents were inhibited by the phenols in a non-competitive antagonistic way.

There are very few studies which demonstrate the relaxant effect of the essential oil of thyme on vascular smooth muscle. In experiments on mice, guinea pigs and rabbits Guseinov found that the essential oil of Kochi Thyme (T. kotchyanus) was non-toxic and produced hypotensive effects in rabbits at concentrations of 1 mg/kg.

Spasmolytic activity of Thymus vulgaris extracts

The therapeutic value of the thyme herb, indeed, depends on the quantity of phenols in the essential oil (see above). Therefore, for all the Thymus species, one has to prefer those that contain a high concentration of thymol and/or carvacrol. However the presence of a non-volatile principle in the Thymus species has always been supposed. This was established by Van den Broucke and Lemli who examined the correlation between the phenol content of Thymus vulgaris liquid extracts and the spasmolytic activity on the muscles of the guinea pig ileum and trachea. The extracts showed a high spasmolytic action, but no correlation between phenol content and activity could be observed. The thymol and carvacrol concentration of extracts was much too low (< 0.001 per cent) and could not be responsible for the antispasmodic activity.

The authors continued their experiments testing flavonoids isolated from Thymus vulgaris () in vitro for their spasmolytic activity on the smooth muscles of the guinea pig ileum and of the rat vas deferens. Both the flavones and the thyme extracts inhibited responses to agonists which stimulate specific receptors (acetylcholine, histamine, noradrenaline) as well as to agents whose actions are not mediated via specific receptors (BaCl2). Flavonoids appeared to act as musculo-tropic agents. Musculotropic spasmolysis is complex and the results of this study could not clarify the events in the muscle completely. However, inhibition of Ca induced contractions in K+ depolarized muscles pointed to a possible decrease in the avaibility of Ca2+. Flavones induced relaxation of the carbachol-contracted tracheal strip without stimulation of the /32-receptors, which were blocked by propranolol. This relaxation could probably be due to an inhibition of the phosphodiesterase, followed by an increase of the intracellular c-AMP level.

The flavonoid pattern of T. satureioides differs from that of the other Thymus species in the high portion of polymethoxylated flavones. The in vitro tracheal relaxant activity of thyme extracts prepared from T. satureioides compared with that of the pure flavones (same quantity of flavones), supports the action of a thyme extract to be almost completely explained by the content on flavones independently from their degree of permethoxylation . The affinity of methoxylated flavones in the guinea pig ileum does not differ significantly from that of luteolin and apigenin. However, methylation of the hydroxy groups of the flavone skeleton increases the relaxant activity.

Further evidence of the spasmolytic effects of flavonoids was provided by Capasso. They screened 13 flavonoids for their effects on contractions in guinea pig ileum induced by prostaglandin E2(PGE2), Leukotriene D4 (LTD4), acetycholine and BaCl2. The flavonoids showed spasmolytic effects that may be due to a non-specific action since they were found to be active against contractions induced by several agents. Flavonoids have also been shown to inhibit electrically-induced contractions (transmural electrical stimulations). Flavonoids inhibit the contractile responses probably through a reduction of calcium influx by way of calcium channels and through inhibition of calcium release from intracellular stores, decreasing the calcium concentration available for contractile machinery .

Spasmolytic activity of extracts of different Thymus species

Blazquez examined the spasmolytic activity of Thymus webbianus and Thymus leptophyllus extracts. Air-dried leaves and stems of these species were extracted with 70 per cent aqueous methanol. The methanol was removed and the remaining aqueous fraction was successively treated with diethyl ether, ethyl acetate and butanol. The results of this study showed that the extracts of both plants have significant spasmolytic effects on isolated rat duodenum. In general, a difference in the responses produced between differently polar extracts was observed. In fact, the diethyl ether extracts (low polarity) produced a dose-dependent reduction in acetylcholine-induced contractions at 1, 10, 100 µg/ml concentrations, whereas the ethyl acetate extracts (middle polarity) and the butanolic (high polarity) were inactive at the same concentrations, but active only at higher concentrations. Thymus webbianus consistently proved to be more active than Thymus leptophyllus. Zafra-Polo) isolated two steroidal compounds with relaxant properties from the diethyl ether extracts of Thymus webbianus. The compounds could be considered as derivatives of stigmastenone and its isomer β-sitostenone.

Blazquez investigated the effects of a diethyl ether extract of the leaves of Thymus leptophyllus on rat uterine and aorta strip muscle. It inhibited the contraction of uterine smooth muscle at lower concentrations than those observed for aorta strips. Rat uterus experiments with and without extracellular calcium yielded similar ED50 values suggesting a non-specific mechanism for the relaxant activity. In the presence of extracellular calcium, the extract inhibited the contractile response of rat aorta induced by K+ depolarising solution, and had a lower inhibitory effect on noradrenaline-induced contraction.

An ethanolic extract of the leaves of Thymus orospedanus has been shown to significantly decrease arterial blood pressure in Wistar rats, at a dose of 150mg/kg during the first hour following the administration, whilst the 300 mg/kg dose continued to show hypotensive effects for up to 5 h. Flavonoids have also been shown to have vasodilator effects in isolated rat and rabbit vascular smooth muscle . This vascular smooth muscle relaxation has been attributed to several mechanisms including decreased transmembrane Ca2+ uptake and inhibitory effects on cAMP and cGMP-phosphodiesterases or on protein kinase C.