Proposed mechanisms of action of herbal drugs and their biologically active constituents in the treatment of coughs: an overview

In vivo

Expectorant effects in vivo were confirmed in some Glycyrrhiza spp. components for an ointment containing menthol and camphene (Kuang et al., 2018; Schäfer & Schäfer, 1981; Schönknecht et al., 2017). In the murine model based on the determination of the phenol red secretion, the Glycyrrhiza spp. components (liquiritin apioside, liquiritin, or liquiritigenin) exerted expectorant activities after a 3 day treatment (50 mg/kg per os). Surprisingly, potent antitussive activities of the same substances were shown as well (see below) (Kuang et al., 2018). Bronchodilatory and secretolytic effects of the insufflated ointment on the basis of menthol, camphene and essential oils (Pinimenthol^®) were observed in the guinea pig model of acetylcholine induced bronchospasms (50% bronchospasm reduction and 44% increase in tracheobronchial secretion). Surprisingly, a higher secretion increase (54%) was found after the application of the ointment on the epilated skin (Schäfer & Schäfer, 1981).

In vitro

Increased secretion of watery mucus by bronchial cells (i.e., secretolytic effects) after administration Hedera saponins (α- or β-hederin, respectively) or Glycyrrhiza spp. (glycyrrhizin) is explained by an indirect β₂-mimetic mode of action. In the case of α-hederin, inhibition of β₂-receptors internalization and increase in β₂-receptor binding were observed. These processes result in an increased intracellular cAMP level, which subsequently elevates surfactant production, leading to a decreased mucus viscosity, and secretion in the alveolar type II cells. This may explain the secretolytic and bronchospasmolytic effect of the aforementioned saponins (Greunke et al., 2015; Sieben et al., 2009).

Menthol stimulated Cl⁻ secretion across canine tracheal epithelium, probably through a Ca²⁺-dependent mechanism. The monoterpene thus may influence mucociliary transport in the respiratory tract (Chiyotani et al., 1994). Reduced secretion of certain mucins (mainly MUC5AC, MUC2, MUC19) was demonstrated, for example, for 1,8-cineole (Sudhoff et al., 2015), glycyrrhizin (Ho-Jin et al., 2006; Li et al., 2018; Nishimoto et al., 2010) and for the fixed combination of the Thymi herba and Primulae radix extracts (Seibel et al., 2018a) in various cell cultures. The reduction in mucin production was dose-dependent, with the effective concentrations in in vitro experiments being roughly in tens to hundreds of µM. The mechanism of action is not fully clear, attenuation of the transcription factor nuclear factor-κB (NF-κB) activity was suggested (Sudhoff et al., 2015). The secretomotoric effect is addressed less often, but is not irrelevant. Glycyrrhetinic acid promoted the differentiation of the nasal epithelial cells by the stimulation of the re-epithelialization of the nasal mucosa and increased the number of cell cilia in the patients suffering from chronic vasomotor rhinitis (Passali et al., 2017). There is also evidence of a secretomotoric effect for certain herbal drugs such as Foeniculi amari fructus, Foeniculi dulcis fructus and Anisi fructus. Notably, some other herbal drugs used in cough therapy had no effect on the mucociliar transport rate (Plantaginis lanceolatae folium and Liquiritiae radix) or caused its inhibition (Althaeae radix) (Müller-Limmroth & Fröhlich, 1980). The effects of Thymus spp. are diverse (Müller-Limmroth & Fröhlich, 1980; Nabissi et al., 2018; Wienkötter et al., 2007). Thymus extract is a complex mixture of essential oil (mainly thymol and carvacrol) and flavonoids (apigenin, luteolin and their glycosides). Bronchospasmolytic effects of thymol are related to its agonism on β₂-receptors (Beer, Lukanov & Sagorchev, 2007), to the blockade of Na⁺ channels (Haeseler et al., 2002) and the activation of the TRPA1 channels (at small thymol doses) (Lee et al., 2008). This latter effect is also exhibited by carvacrol (Xu et al., 2006). Additionally, apigenin, luteolin and its glycosides were identified as possible spasmolytic compounds (Van Den Broucke & Lemli, 1983). Last but not least, Thymi herba extract inhibited ex vivo endothelin-induced contraction of isolated rat trachea smooth muscles via non-competitive interaction with endothelin receptors. However, thymol was found to be an inactive extract component (Engelbertz et al., 2008).

Notably, stimulation of respiratory bitter taste receptors (TAS2R) also leads to bronchodilation (Liggett, 2013). Since various natural compounds are TAS2R agonists (Meyerhof et al., 2010), this effect could represent another mechanism of their action. Also noteworthy, the essential oils produce other important effects, including anti-inflammatory and antibacterial (see below).

Clinical evidence

1,8-Cineole (eucalyptol), used to relieve nasal congestion in the patients with common cold, is the principal constituent of the essential oil of eucalyptus. The oil probably exhibits expectorant and mucolytic effects (Dixit, Rohilla & Singh, 2012) in the bronchial epithelium. Administration of 1,8-cineole to asthmatic patients resulted in a substantially improved lung function and alleviation of dyspnoea (Worth & Dethlefsen, 2012). 1,8-cineole also alleviated the discomfort of non-purulent rhinosinusitis in the patients with acute rhinosinusitis (Kehrl, Sonnemann & Dethlefsen, 2004).

Interesting clinical study (randomized, double-blind controlled) dealt with the effect of a spray containing the essential oils of Eucalyptus citriodora, E. globulus, Mentha × piperita, Origanum syriacum, Rosmarinus officinalis, and a placebo spray (application five times a day over 3 days). Compared to those from the placebo group (P = 0.019), the members of the study group felt a more pronounced relief from the symptoms that included a sore throat, hoarseness, or cough, after a 20 min period. In 3 days, however, the differences in the intensity of the symptoms were negligible (P = 0.042) (Ben-Arye et al., 2011).

As regards the efficiency of Hedera helix leaf extracts as an expectorant, the clinical data available are not conclusive. According to a review summarizing data from 13 clinical trials, herbal preparations from H. helix could serve as a remedy for early symptoms of respiratory tract infections, regardless of age. Sufficient clinical evidence has been gathered to prove the positive effects of ivy preparations in cough therapy. However, the amount of data on an improvement of cough expectoration, night coughing and cough-associated sleep disruption is insufficient (Barnes et al., 2020). Interestingly, yet another study reported that Hederae folium extract could serve as an alternative to acetylcysteine in the improvement of the respiratory function in both children and adults. In both groups, alleviation of all symptoms was recorded after 7 days. Notably, a more pronounced improvement in the number of cough attacks and cough-related sleeping disorders was observed in the participants taking the Hedera syrup (Kruttschnitt et al., 2020). Effectiveness and safety of dry Hedera helix L. extract was also studied in a non-randomized, non-interventional, multicenter, open-label, post-authorization effectiveness study (464 patients aged 2–12 years with productive cough). Significant improvement in several parameters (cough, chest pain, wheezing, dyspnoea, auscultation changes or body temperature) and no adverse drug reactions were reported (Schönknecht et al., 2017). Yet another systematic review, focused on the use of Hederae folium extracts for the treatment of acute upper respiratory tract infections, confirmed their safety as cough medications. By contrast, as regards minimizing cough severity and/or frequency, the clinical significance of the data seemed very poor (Sierocinski, Holzinger & Chenot, 2021). Finally, a retrospective, multicenter cohort study of the expectorant properties of the Hedera helix leaf extracts in pregnant women revealed their safety for the fetus (Alkattan et al., 2021).

As regards the preparations from thyme, children 2 months to 14 years old suffering from a bronchial catarrh or bronchitis were given a dose of a thyme syrup over the period of 7–14 days in an open study. Alleviation of coughing was recorded in 94% of the patients (European Scientific Cooperative on Phytotherapy, 2003). By contrast, another study (randomized, double-blind) dealt with patients with a productive cough, accompanying uncomplicated respiratory infections. A thyme syrup or a bromhexine preparation was administered to the patients for 5 days, but no appreciable difference between the two interventions was reported (Knols, Stal & Van Ree, 1994). While these studies are rather unconvincing in terms of clinical efficacy, long-term use of Thymus spp. is supportive of the application of various products from this plant, including the plant material, extracts and the essential oil as traditional herbal remedies for the cold-accompanying productive cough. Thyme components were also employed as components of fixed combination with those from other plant species. Specifically, a fixed combination of Thymi herba and Primulae radix extract reduced the Bronchitis Severity Score (BSS) in acute bronchitis, both in adults and children (Gruenwald, Graubaum & Busch, 2005, 2006; Kemmerich, 2007; Ludwig, Stier & Weykam, 2016).

Next, just a single study on licorice revealed that licorice pastille significantly decreased the Leicester Cough Questionnaire scores of the patients with a chronic cough (Ghaemi et al., 2020).

More data are available on fennel. Fennel seeds facilitate the transport of extraneous particles via the enhancement of the ciliary motility of the respiratory apparatus. The inhalation of fennel essential oil stimulates the smooth muscle contraction of the trachea and, consequently, a smoother expectoration of the mucus, bacteria, and other foreign corpuscles occurs. trans-Anethole, the principal constituent of the oil, is also analogous to catecholamines, namely epinephrine, norepinephrine, and dopamine. It is therefore not surprising that various sympathomimetic effects of fennel, including a bronchodilatory activity, were observed (Javed et al., 2020). The efficacy of Zataria multiflora and Foeniculum vulgare essential oils (Tussivin^® oral drops) in comparison to clobutinol and placebo in the treatment of acute cough in 119 patients was reported in a clinical study. The dose of 20 drops of each preparation three times a day over 3 days was administered to the patients, aged over 14. Undisputable recovery in the patients suffering from a long-term cough, lasting for more than 7 days, was observed when Tussivin^® oral drops were used (Afzali, Kashanian & Akbari, 1998; Mahboubi, 2018).

Finally, studies of the effect of aniseed (Anisi fructus, Pimpinella anisum) in humans are rare. The effect of the water extract from the seeds was examined in 26 patients with a chronic rhinosinusitis with no polyps (200 μg/nostril vs one fluticasone dose/nostril; in 12 h intervals for 4 weeks). Both preparations alleviated the symptoms of mucosa inflammation and sinonasal disorders. In comparison to fluticasone, anise caused a more pronounced improvement of the scores for both the sinonasal outcomes and rhinological symptoms (Singletary, 2022).

In vivo

Among the antitussive medicinal plants studied with the in vivo cough models, Althaea officinalis is the most frequently reported. Its cough suppressing action could be, in principle, based on the mucilaginous polysaccharides, mainly rhamnogalacturonans (Nosáľvá et al., 1992, 1993; Šutovská et al., 2011, 2007, 2009). The antitussive effects were also documented for Plantago lanceolata and Pelargonium sidoides in the form of extracts (Bao et al., 2015; Boskabady et al., 2006). Certain effects were reported for some coumarins and flavonoids (Huang et al., 2020). Limited amount of data on the decrease in cilia beating and mucus clearance are available (inhibition for Althaeae radix (Müller-Limmroth & Fröhlich, 1980) and stimulation for Pelargonium sidoides root extract (Neugebauer et al., 2005)). Surprisingly, significant antitussive effects have been described also for Glycyrrhiza spp. constituents (liquiritin apioside, liquiritin, and liquiritigenin at 50 mg/kg per os) in the murine model of cough, induced by ammonia. These effects were partially antagonized by pretreatment with a 5-HT receptor antagonist (methysergide) or with a K_ATP channel blocker (glibenclamide), which implies the participation of these structures (Kuang et al., 2018).

In vitro

In the in vitro studies, a moderate bioadhesion to the porcine buccal epithelial tissue has been confirmed for the mucilaginous polysaccharides from Althaea officinalis and Plantago lanceolata, and the effects were concentration-dependent in both cases (Schmidgall & Hensel, 2002; Schmidgall, Schnetz & Hensel, 2000). Importantly, there are differences among the cell lines. The polysaccharides were actively taken up by the epithelial cells and could thus exhibit intracellular effects, as found for those from Althaea officinalis. By contrast, fibroblasts in the same experiment simply absorbed the polysaccharides on their surface, and no cellular internalization of the polymers was observed (Deters et al., 2010).

Clinical evidence

The only clinical studies that involved syrup preparations combining more herbal components (Althaea officinalis, Cetraria islandica, Plantago lanceolata and Pelargonium sidoides) were published recently. Therefore, a synergistic action of individual components should be anticipated. Relevant molecules in the mixtures (e.g., mucilage, iridoids, flavonoids, phenylethanoids, phenolic acids) act in complexes, exhibiting their effects through diverse mechanisms. While each individual component contributes to clinical evidence, the result is not just a simple sum of individual effects. No product tested was a traditional herbal medicinal product. Thus, the preparations were planned to be marketed under the medical device legislation (as opposed to the European medicinal products for human use legislation), which also significantly influenced the conclusions of these studies.

The following plants are separately used in various preparations in European official phytotherapy: Althaea officinalis (EMA, 2016), Cetraria islandica (EMA, 2014a), Plantago lanceolata (EMA, 2014b) and Pelargonium sidoides (EMA, 2018a), and categorized as “traditional use”. With the exception of Pelargonium sidoides, no reliable clinical studies have been performed for the others, with the registration of the corresponding single-component herbal products being allowed by the pertinent legislation.

Besides the clinical trials published up to and including 2018, summarized in the Assessment Report (EMA, 2018b), some more recent data for Pelargonium sidoides are available. Martin et al. (2020) found out that the extract from Pelargonium sidoides root helped to significantly reduce the need for concomitant antibiotic prescriptions, and substantially shortened the time of patient recovery. The results were published in a retrospective cohort study in 117,182 patients 20–60 years of age. In another interesting study, the effect of the extract from the roots of P. sidoides on the immune response of athletes was investigated by Luna et al. (2011). The extract enhanced the production of immunoglobulin A in the saliva and reduced the levels of interleukin (IL) IL-15 in the nasal mucosa and that of IL-16 in the serum (Luna et al., 2011). Riley et al. (2019), administered 40 mg EPs^® 7630 or placebo three times a day for 10 consecutive days to a set of 105 patients (men and women 18 to 55 years of age) with common cold symptoms (nasal drainage, sneezing, sore throat and nasal congestion, hoarseness, muscle aches, headache, scratchy throat, cough, or fever). Compared to the placebo group, EPs^® 7630 alleviated cold symptoms in the other group (Riley et al., 2019). Similarly, the effects of P. sidoides extract EPs^® 7630 in the treatment of acute respiratory tract infections (RTI) in children was reviewed by Careddu & Pettenazzo (2018). A significant improvement in the intensity of RTI symptoms, excellent safety and tolerability of EPs^® 7630 was evidenced by all clinical studies (Careddu & Pettenazzo, 2018). Notably, the potency of EPs^® 7630 in treating human coronavirus infections (HCoV) was also investigated. A set of 61 patients, out of which 37.7% were infected with a HCoV strain, and had the symptoms of common cold with an accompanying viral infection, were given 20 mg of EPs^® 7630 three times a day. A significant improvement of the cold symptoms, especially in the HCoV group, was observed. A complete recovery or significant alleviation of the symptoms was recorded in 80% of the patients. In addition, the majority of the patients reported satisfaction with EPs^® 7630 treatment (Keck et al., 2021). A recent meta-analysis summarized the data from seven clinical trials with a total of 1,099 participants. While adult patients had the symptoms of common cold, the child group also included those suffering from an acute non-streptococcal tonsillopharyngitis. Both groups reported a sore throat and hoarseness. Again, alleviation of both symptoms and faster recovery in the EPs^® 7630 treated groups vs the placebo was reported (Kamin et al., 2022). Finally, according to Seifert et al. (2019), the use of EPs^® 7630 reduced the symptoms, decreased the need for parallel administration of paracetamol, and helped to achieve a faster recovery, based on reviewing six clinical trials with 523 children aged 6 to 10 years with acute bronchitis or non-streptococcal tonsillopharyngitis.

In vivo

Of the expectorant plants studied in vivo, the Hedera helix in the form of extract or its fraction has probably been the most frequently investigated. Its anti-inflammatory effects were shown in the ALI model (Shokry et al., 2022a) as well as in the model of arthritis (Shokry et al., 2022b). The effective doses were roughly 100–200 mg/kg. The same effects were shown for its principal component, hederacoside C (5–50 mg/kg i.v. or i.p.) (Akhtar et al., 2019; Han et al., 2022). trans-Anethole, the principal component of Anisi aetheroleum or Foeniculi amari fructus aetheroleum, also exhibited anti-inflammatory action in the ALI and COPD models in the effective doses ranging from 62.5 to 500 mg/kg i.p. or orally (Kim, Lee & Seol, 2017). Besides the respiratory system, trans-anethole was effective in the paw edema, pleurisy or ear edema models of inflammation in similar dose ranges (Domiciano et al., 2013; Wisniewski-Rebecca et al., 2015). 1,8-Cineole, the principal component of Eucalypti aetheroleum, ameliorated inflammatory changes in three different models of the lung injury–the ALI, allergic asthma and COPD. Inhibition of the inflammatory cell infiltration was observed in all cases. The treatment was oral (up to 260 mg/kg) or by inhalation (10 mg/mL for 30 min, 6 h before the allergic challenge). In the case of the COPD model, the administration was repeated over 12 weeks (Lee et al., 2016; Yu et al., 2018; Zhao et al., 2014). Importantly, in both long-term respiratory pathologies (asthma and COPD), restoration of the impaired lung functions was confirmed (Lee et al., 2016; Yu et al., 2018). Anti-inflammatory properties were also reported for the licorice root extract and its main components, glycyrrhizin and liquiritigenin. Attenuated mucus hyperproduction and goblet cell hyperplasia were shown after glycyrrhizin subcutaneous administration in two different murine models of lung inflammation (intratracheal instillation of LPS or of IL-4). These effects were at least partially mediated by the inhibition of the MUC5AC gene transcription (Nishimoto et al., 2010). A mixture of Thymus and Primula extracts had similar effects in a rat model of inflammation in tens to roughly 400 mg/kg given orally in one single dose or three consecutive doses (Seibel et al., 2018a, 2018b). Apart from the respiratory system, the licorice root extract and liquiritigenin had positive effects on the murine liver inflammation (Yu et al., 2015) and rat paw edema (Kim et al., 2008), respectively. The doses of liquiritigenin were 15 mg/kg given intravenously for 2 days or 50 mg/kg orally for 3 days, and that of the extract 300 mg/kg orally for 3 days.

Among the antitussive plants, anti-inflammatory in vivo effects on the airways were reported for the extracts from Pelargonium sidoides (EPs^® 7630) and from Althaea officinalis. In both cases, the effective doses were roughly 50–100 mg/kg. Positive changes were observed in the tracheal tissue and were dose-dependent, even though the way of administration was different (orally or by inhalation) (Bao et al., 2015; Kolodziej, 2011; Mikaili et al., 2010). As regards pure substances, scopoletin and plantamajoside showed anti-inflammatory properties in murine models of lung injury (both administered i.p. at the doses between 10–100 mg/kg) (Leema & Tamizhselvi, 2018; Wu et al., 2016). Besides the airways, positive effects on wound healing and pain were observed together with an increase in the phagocytic activity of the macrophages or decrease in the leukocyte infiltration. In these aspects, the studies mostly showed the effect of the Althaea officinalis extract. Some data on the extracts from Cetraria islandica and Plantago spp. are also available. Both the ways of administration (orally, i.p., s.c. or topical) and the effective doses (from 2.5 to 200 mg/kg) in these experiments were variable (Freysdottir et al., 2008; Marchesan et al., 1998; Rezaei et al., 2015; Shipochliev, Dimitrov & Aleksandrova, 1981; Valizadeh et al., 2015; Yazdian & Irani, 2015). From pure substances, anti-inflammatory effects in other tissues were reported for scopoletin (5 and 10 mg/kg, i.p.) in the murine model of the paw edema (reduced pain and number of writhing) (Chang et al., 2012).

In vitro

The hederacoside C (0.5–5 μg/mL) was shown to decrease the expression of the TLR2 and TLR4 receptors in vitro elevated by the Staphylococcus aureus infection. Since these receptors recognize the microbial noxious stimuli, their downregulation may modify the extent of the immune response (Akhtar et al., 2019). Liquiritigenin and isoliquiritigenin, components of licorice, suppressed eotaxin secretion in the human fetal lung fibroblast. It is therefore possible that the migration of the inflammatory cells may be reduced (Jayaprakasam et al., 2009). trans-Anethole protected against decreased cell viability, cell barrier injury and increased cell apoptosis, albeit at very high concentrations of 3 and 5 mM. Although these effects were observed in the rat intestinal epithelial cells, the respiratory epithelial cells may respond in a similar manner. Importantly, changes in the expression of 493 genes, most of which were related to antigen processing and presentation, complement and coagulation cascades, and to the NF-κB signaling (see Subsection 5.4.4.), were detected (Yu et al., 2022). The stimulation of the macrophage phagocytic activity and migration, and the inhibition of complement activation was reported for the Althaea officinalis extract and its polysaccharide fraction (WHO, 2002; Scheffer, Wagner & Proksch, 1991; Bonaterra et al., 2020). The same was described for Cetraria islandica products (100 μg/mL) studied in the granulocytes (Ingolfsdottir et al., 1994; Olafsdottir et al., 1999; Shrestha, Clair & O’Neill, 2015). Anti-complement activity was also found in some flavonoids, including kaempferol, kaempferol-3-O-rutinoside and quercetin (Huang et al., 2020).

Clinical evidence

The anti-inflammatory, mucolytic, and bronchodilatory effects were confirmed for 1,8-cineole (200 mg/three doses daily/6 months as a supplementary therapy during winter) in a double-blind, placebo-controlled multi-center-study with 242 patients. Statistically significant improvement of multiple criteria was observed (frequency, duration and severity of exacerbations, lung function, respiratory symptoms, quality of life) (Worth, Schacher & Dethlefsen, 2009).

Although the cough is the main objective of this article, the anti-inflammatory effects not directly related to the respiratory system or cough deserve a very short mentioning for the sake of completeness. Positive anti-inflammatory effects of quercetin were found in various clinical trials addressing e.g., rheumatoid arthritis (Javadi et al., 2017), polycystic ovary syndrome (Vaez et al., 2023) or hemodialysis patients (Castilla et al., 2006). Some effects were also reported for trans-anethole (Hamada et al., 1999), limonene (Ostan et al., 2016; d’Alessio et al., 2013), luteolin (Casetti et al., 2009; Lunardelli et al., 2019), combination of luteolin, quercetin, and rutin (Taliou et al., 2013), hydroalcoholic extract of Glycyrrhiza spp. root (Zabihi et al., 2023) and glycyrrhizic acid (Cao et al., 2020). In contrast, other studies reported negligible or no effects on inflammation (Sajadi Hezaveh et al., 2019; McAnulty et al., 2008; Heinz et al., 2010; McAnulty et al., 2013; Hodgin et al., 2021; Dehghani et al., 2021).

Experimental methods

The same in vivo models of inflammation are used as in the research of inflammation mentioned above (ALI, lung infection etc.). The inflammatory models outside the respiratory system are also relatively common. The potent activators of inflammation, mainly IL-1, IL-6 and TNF-α, are the most often studied cytokines. The interpretation of the obtained results is, at least at first glance, somewhat contradictory. While the suppression of the pro-inflammatory cytokine production is regarded as a protection against an excessive inflammation and tissue damage, their production is, in turn, interpreted as a stimulation of the immune response. For in vitro studies, various cell models are used, but not all of them are related to the airways. To induce the inflammatory conditions, LPS is usually applied, while other stimuli, such as histamine, INF-γ or SARS-CoV-2 are scarce (Li et al., 2018; Papies et al., 2021; Vigo et al., 2005).

Pharmacological studies on the effects on the cytokines

In vivo

The in vivo suppression of the pro-inflammatory cytokine production was demonstrated for both the Hedera helix extract and its phenolic rich fraction in a murine model of the ALI and in a rat model of arthritis (doses ranging 100–200 mg/kg, in all cases orally administered) (Shokry et al., 2022a; 2022b). Hederacoside C (5–50 mg/kg i.v. or i.p.) had similar effects in two different murine models of inflammation (ALI or inflammation induced by Staphylococcus aureus infection) (Akhtar et al., 2019; Han et al., 2022). Similarly, trans-anethole (125–250 mg/kg, i.p. and orally, respectively) protected against the increase in the pro-inflammatory cytokine levels in BALF in the murine model of ALI (TNF-α, IL-1β and IL-6) and in the COPD model (TNF-α and IL-6) (Kim, Lee & Seol, 2017; Kang et al., 2013). In the former case, the serum cytokine levels were reduced as well. The same was observed in the rat model of pleurisy for oral doses of trans-anethole of 250 and 500 mg/kg (Domiciano et al., 2013). The licorice extract given orally in the dose of 300 mg/kg in the murine liver inflammation model and menthone administered by inhalation (0.3 up to 1.2 mg/L at 2.8 mL/min for 25 min) in the asthma model displayed similar effects (Yu et al., 2015; Su & Lin, 2022). Analogously, 1,8-cineole decreased the levels of the principal pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in three different models of inflammation (ALI, asthma and COPD models). Importantly, the levels of some anti-inflammatory cytokines (IL-10, IL-4, IL-13) also decreased, which is more supportive of an immunomodulatory effect of the monoterpenoid with the concomitant fine tuning of the inflammatory response (Lee et al., 2016; Yu et al., 2018; Zhao et al., 2014).

The Plantago lanceolata is probably the most frequently reported antitussive plant in animal studies, investigating the effect on the cytokine production. Its components, plantamajoside and luteolin reduced the pro-inflammatory cytokine levels in the murine and rat inflammatory models (ALI or LPS-induced bronchopneumonia) in the effective doses of 20–100 mg/kg i.p. and orally, respectively. The effects of plantamajoside may be mediated by the depression of the lung TLR4 receptors (Wu et al., 2016). On the other hand, the in vivo effects of luteolin were accompanied by a reduced miRNA-132 expression in the bronchial tissue. The authors speculated that this small non-coding RNA molecule might act as a mediator for luteolin in its regulation of the LPS-induced inflammation (Liu & Meng, 2018). Surprisingly, luteolin reduced the amount of the pro-inflammatory cytokines and exhibited anti-inflammatory effects even upon topical application in rat granuloma and murine ear edema models. These effects were enhanced in the presence of the polysaccharides from Citrus grandis Osbeck, suggesting possible potentiation (Chen et al., 2018). The reduction of the pro-inflammatory cytokine levels (IL-1β and TNF-α) was also reported for scopoletin and β-glucan administration in similar doses (1–10 mg/kg i.p., and 50 mg/kg orally, respectively) (Leema & Tamizhselvi, 2018; Chang et al., 2012; Babayigit et al., 2005; Bedirli et al., 2007; Senoglu et al., 2009). In the case of scopoletin, the modification of the NF-κB signaling was suggested. In contrast to previous studies, an increase in the levels of the pro-inflammatory and anti-inflammatory cytokines after luteolin administration was reported. However, an atypical model of airway inflammation (murine tuberculosis) was used in this study. Regardless of it, the effects of luteolin were considered positive due to the reduced level of necrosis and enhanced long-term protection against tuberculosis mediated by the T and NK cells (Singh et al., 2021).

In vitro

In accordance with the in vivo studies, decreased production of the pro-inflammatory cytokines was found after the incubation with several pure components of the expectorant plants, such as hederacoside C (Han et al., 2022), trans-anethole (Chainy et al., 2000), 1,8-cineole (Juergens, Stöber & Vetter, 1998), liquiritigenin (Kim et al., 2008), glycyrrhizic acid (Li et al., 2018), liquiritin (Yu et al., 2015), and three secondary metabolites from Marrubium vulgare (Shaheen et al., 2014). These results were obtained on diverse cell lines (RAW264.7, J774A.1, THP-1) as well as on the monocytes and lymphocytes from healthy human donors or on the human nasal epithelial cells. The effective concentrations in all cases ranged from 20 to 100 µM. The effects on the cytokine production were dose-dependent and corresponded to the inhibition of mRNA expression (if determined). The dose-dependent decreasing effects on the anti-inflammatory cytokine production were also shown for many antitussive plant components. Among them, the plantamajoside (Wu et al., 2016; Liu et al., 2019; Ma & Ma, 2018; Son et al., 2017), luteolin (Chen et al., 2018) and scopoletin (Cheng, Cheng & Chang, 2012) were the most frequently reported. In all experiments on diverse cell lines, the effective concentrations were roughly in the order of tens of μg/mL. Polysaccharide lichenan increased the secretion of anti-inflammatory IL-10 compared to the IL-12p40, the polymer may thus rather tune the inflammatory response analogously to 1,8-cineole mentioned above (Freysdottir et al., 2008). The effects on the cytokine production were also found for various plant extracts such as Althaea spp. (Scheffer, Wagner & Proksch, 1991), Pelargonium sidoides (EPs^® 7630) (Papies et al., 2021; Kolodziej et al., 2003; Witte et al., 2020), Cetraria islandica (Freysdottir et al., 2008; Omarsdottir, Olafsdottir & Freysdottir, 2006) (effective concentrations roughly 50–500 μg/mL) and for β-glucans (Murphy et al., 2020).

Discussion

Many in vivo and in vitro studies reported the possible effects of the cough phytotherapy on the cytokines and their signaling pathways. As mentioned above, the experimental model strongly influences the results. As demonstrated by these examples, the selection of the cell line is crucial. (1) The activated monocytes responded differently to the Althaea sp. extracts from the differentiated macrophages or the RAW264.6 cells. Depending on the experimental setup, enhancing, neutral, and decreasing effects on the cytokine production were found (Kim, Lee & Seol, 2017; Scheffer, Wagner & Proksch, 1991). (2) The epithelial cells and fibroblasts responded to the polysaccharides from Althaea officinalis in very different ways (Deters et al., 2010). Although the studies are hardly comparable, (3) the mechanisms modifying the cytokine production are probably very diverse. For example, trans-anethole blocks the I-κBα kinase, and thus subsequently decreases the I-κBα (nuclear factor of the kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) degradation (Chainy et al., 2000), plantamajoside decreased the expression of the TLR4 receptors (Wu et al., 2016), scopoletin was found to down-regulate the cytokine transcription factors GATA-3 and the nuclear factor of the activated T cells (NFAT) with the participation of the protein kinase C (Cheng, Cheng & Chang, 2012). The effects of the lichenan may be mediated by DC-SIGN, which is a C-type lectin receptor present on the surface of the dendritic cells and macrophages. For the lichenan, tolerogenic effects similar to those of vitamin D₃ were hypothesized (Freysdottir et al., 2008). In addition, experiments often focus on just one possible mechanism of action, but a substance can act in multiple ways.

Experimental methods

The same inflammation models used in experiments on the cytokines mentioned previously (ALI, paw edema etc.), are employed for in vivo research. For in vitro studies, diverse cell models of inflammation have been developed based on the measurement of cyclooxygenase activity or expression of the related genes. Special biochemical assays are also available. The inducible COX-2 was more frequently investigated than the house-keeping COX-1 isoform. The effects on 5-lipoxygenase were studied using special immune assays.

Pharmacological studies on the effects on cyclooxygenase and lipoxygenase

In vivo

In the murine model of ALI, administration of the ethanolic Hedera helix leaf extract and the phenolic rich fraction (orally 200 and 100 mg/kg, respectively) downregulated the expression of COX-2 (Shokry et al., 2022a). Scopoletin, constituent of the Pelargonii radix, decreased induced expressions of COX-2 and iNOS in the murine λ-carrageenan model of the paw edema (Chang et al., 2012). A fixed combination of the thyme herb and primula root extracts reduced the levels of LTB₄, cysteinyl leukotrienes and PGE₂ in BALF in a rat model of the LPS-induced pulmonary inflammation (Seibel et al., 2018b).

In vitro

The ethanolic extract from Hedera helix leaves and its phenolic rich fraction inhibited human recombinant COX-2 activity measured by a commercial colorimetric inhibitor screening assay kit. The latter was slightly more effective (Shokry et al., 2022a). Inhibition of both COX isoenzymes was reported for various compounds isolated from Marrubium vulgare (Neamah, Sarhan & Al-Shaye’a, 2018; Sahpaz et al., 2002) and for carvacrol, a monoterpene phenolic constituent of the essential oil from Thymus vulgaris (Landa et al., 2009). Among antitussives, a hydroalcoholic extract from Plantago lanceolata inhibited the COX-2 activity, but not that of lipoxygenase in cell-free systems (Herold et al., 2003). Accordingly, plantamajoside decreased the PGE₂ levels in the human gingival fibroblasts, stimulated by LPS (Liu et al., 2019). On the other hand, no effects of the Plantago lanceolata extract on the elevated COX-2 mRNA and the PGE₂ levels were detected in the LPS/INF-γ stimulated murine macrophages (Vigo et al., 2005). Interestingly, Althaea rosea flower extract upregulated the expression of COX-2 and iNOS in the RAW264.6 cells, and these effects were interpreted as immunostimulation (Kim, Lee & Seol, 2017).

Inhibition of lipoxygenase was reported for the primula extract, and for various components of the thyme extract; synergistic effects of all compounds are possible. The same was observed for a mixture of dry extracts from the thyme herb and the primula root in the rat whole blood and in the human monocytes and neutrophils stimulated with Ca²⁺-ionophore (IC₅₀ roughly units to low tens of µg/mL) (Seibel et al., 2018b).

Discussion

There are apparent differences in selectivity among the active components in relationship to the inhibition of the cyclooxygenases even from one plant. For example, luteolin-7-O-β-glucopyranoside, oleanolic acid and luteolin-7-O-rutinoside from Marrubium vulgare inhibited preferably COX-1, while apigenin-7-O-β-glucopyranoside, luteolin-7-O-rutinoside and rosmarinic acid inhibited COX-2 (Neamah, 2018). Similarly, COX inhibition was reported for various phenylpropanoid esters from Marrubium vulgare and three of them, acteoside, forsythoside B, and arenarioside preferably inhibited the COX-2 (Sahpaz et al., 2002).

Experimental methods

Elevations of the serum superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), or decreases of the malondialdehyde (MDA) and myeloperoxidase (MPO) levels are most frequently measured variables for the antioxidant effect quantification. The MPO serves not only as a marker of oxidative stress, but also as that of neutrophil accumulation in the inflammatory tissue. In in vitro studies, various additional methods for the evaluation of the antioxidant properties were applied, including the measurement of the total-oxidant capacity, scavenging effects, ferric-reducing antioxidant power, modulation of the respiratory burst, and others. The results should be treated with caution, as they may easily lead to misleading conclusions. For example, an increase in the levels of antioxidant enzymes may be an adaptor response to the oxidative stress, and ferric reduction can be associated with pro-oxidation (Dodson, Castro-Portuguez & Zhang, 2019; Chen et al., 2015; Macáková et al., 2012).

Pharmacological studies on the antioxidant effects

In vivo

Antioxidant effects or prevention of oxidative stress were found for hederacoside C and 1,8-cineole in the doses of roughly units to tens of mg/mL, i.p. and orally, respectively (Akhtar et al., 2019; Zhao et al., 2014), and for the Hedera helix extracts administered orally in the doses of 100 and 200 mg/kg (Shokry et al., 2022a). Among antitussive plants, the antioxidative effects were found for scopoletin (Leema & Tamizhselvi, 2018), for a secondary metabolite from Cetraria islandica (de Barros Alves et al., 2014), for β-glucans (Bedirli et al., 2007; Gulmen et al., 2010), and for the extract from Pelargonium sidoides (EPs^® 7630) (Bao et al., 2015).

In vitro

In in vitro experiments, the antioxidant effects were observed mainly for licorice and its constituents. This antioxidant activity is attributed to the phenolic content of licorice, with flavonoids and isoflavones being the main candidates as the active substances (Yu et al., 2015; Kim et al., 2008; Racková et al., 2007; Sharma & Pandey, 2013; Singh, Pal & Darokar, 2015). An antioxidant activity was also reported for the extract from Primula veris. Notably, higher antioxidant effects were found in the flowers rather than the roots (both in fresh and dried extracts), and their intensity was directly proportional to the total phenolic content (Tarapatskyy et al., 2021). Among antitussives, Plantago lanceolata and its principal constituent, plantamajoside, probably have certain antioxidant properties as well (Herold et al., 2003; Choi et al., 2008). The same is true for the Althaea officinalis root extract and fraxetin (a compound of Pelargonii radix) (Bonaterra et al., 2020; Chang et al., 1996).

Discussion

Some protective antioxidant effects of the cough herbal drugs or their components were experimentally shown. In animal models, the effective doses were roughly in the order of tens of mg/kg given i.p. or orally. In one study, intramuscular administration of β-glucan in two doses of 2 mg/kg decreased the lung MPO activity in the rat inflammation model (Bedirli et al., 2007). The mechanism of the antioxidative action is not very clear, and probably involves various intracellular targets. In the case of the Hedera helix extracts, the increased expressions of p38-MAPK (mitogen-activated protein kinase) and phospho-p38-MAPK were attenuated, which implies the participation of this signaling pathway (Shokry et al., 2022a). Glycyrrhizic acid, liquiritin and liquiritigenin inhibited the LPS-stimulated NO production in a dose-dependent manner (Yu et al., 2015). Accordingly, liquiritigenin was found to prevent the iNOS induction, albeit at very high concentrations (3–30 mM) (Kim et al., 2008). Three secondary metabolites of Marrubium vulgare had similar effects (Shaheen et al., 2014). The antioxidant activity of Plantago lanceolata is also hypothesized to be mediated by NO-scavenging or by the inhibition of the iNOS gene expression (Vigo et al., 2005). The effects of glabridin, isoflavan from the licorice root were mediated by the elevated expressions of various genes involved in the response to the oxidative stress. Notably, glabridin had antioxidant effects at low concentrations, while at higher concentrations, this isoflavan induced the generation of intracellular ROS and NO (Singh, Pal & Darokar, 2015). Hence, the antioxidant effects are manifested only within a certain dose or concentration ranges, and can be reversed to prooxidant effects at higher concentrations.

Unfortunately, the data on the plant polysaccharides and their antioxidant effects in the cough treatment are scarce. The effects of the orally administered β-glucans outside the airways were reported (Senoglu et al., 2009). However, it is well known that the polysaccharide composition, conformation and molecular weight influence the antioxidant activity. Antioxidant properties become more pronounced with increased content of phenolic acids and proteins (polysaccharide-polyphenol and polysaccharide-protein covalent conjugates and non-covalent complexes), with selenylation and derivatization, such as sulfation, carboxylation, phosphorylation, benzoylation, acetylation or oxidation. Chemical modification is often accompanied by a decrease in molecular weight which is also regarded as positive for the antioxidant potential (Nemzer et al., 2019).

Experimental methods

A possible suppression of the NF-κB signaling by cough phytotherapy was investigated in the same animal inflammation models as those described above. The in vitro experiments on cell cultures were more frequent. From the parameters reported, the amount of the NF-κB and the I-κBα proteins, translocation of the NF-κB into the nucleus, expressions of the related genes and the related protein levels are the most common.

Pharmacological studies on the effects on the NF-κB signaling

In vivo

In two different murine models of airways inflammation (ALI or Staphylococcus aureus infection), the stimulation of the NF-κB signaling was diminished after the administration of hederacoside C (doses of roughly tens of mg/kg, i.v. and i.p., respectively). Importantly, the up-regulated levels of phosphatidylinositol 4,5-bisphosphate (PIP₂), diacylglycerol and inositol-1,4,5-trisphosphate (IP₃) and down-regulated level of PLCγ2 were subsequently also modified, indicating a participation of this signaling pathway (Akhtar et al., 2019; Han et al., 2022). Similar effects were observed for 1,8-cineole in oral doses of 10–100 mg/kg in the murine model of ALI (Zhao et al., 2014). From antitussives, a decrease in the NF-κB signaling was produced by i.p. plantamajoside, luteolin and scopoletin in the inflamed lungs (Wu et al., 2016), and in the model of the cerulein-induced acute pancreatitis linked to a lung injury. Effective doses were roughly in the order of tens of mg/kg (Liu & Meng, 2018).

In vitro

Suppression of the stimulated NF-κB signaling in vitro was observed for the expectorant hederacoside C (Akhtar et al., 2019; Han et al., 2022), trans-anethol (Yu et al., 2022; Aggarwal & Shishodia, 2004; Sen, Traber & Packer, 1996), 1,8-cineole (Greiner et al., 2013), glycyrrhizin (Li et al., 2018) and liquiritigenin (Kim et al., 2008) in diverse cell cultures. The same was reported for active constituents of Plantago spp.: plantamajoside (Wu et al., 2016; Liu et al., 2019; Ma & Ma, 2018; Son et al., 2017; Han, Nam & Lee, 2016), luteolin (Liu & Meng, 2018; Chen et al., 2018) and apigenin (Zhang et al., 2014), and for the coumarins from Pelargonii radix: scopoletin and fraxetin (Hassanein et al., 2020). In all these experiments on the cell cultures, the effective concentrations were roughly in the order of units of μg/mL or tens of μM. The effects of trans-anethole may be mediated by the TLR4 receptor (reported in rat intestinal epithelial cells) (Yu et al., 2022).

Discussion

In vivo suppression of the NF-κB signaling is produced in the doses roughly of tens of mg/kg, regardless of the way of administration. While these effects are evident, their mechanism remains less clear. Modulations of the NF-κB/p65 and I-κBα levels and localizations are probably crucial. For example, glycyrrhizin inhibited histamine-induced mRNA expression and secretion of NF-κB/p65 and I-κBα in the human nasal epithelial cells (Li et al., 2018). Similarly, hederacoside C suppressed NF-κB/p65 and I-κB-α expressions in the RAW264.7 cells exposed to Staphylococcus aureus (Akhtar et al., 2019), while the substance prevented the translocation of NF-κB/p65 into the nucleus in the LPS-stimulated macrophages (Han et al., 2022). A total of 1,8-cineole exhibited similar effects in the human peripheral blood mononuclear cells, in the human glioblastoma U373 cells and the cervical cancer HeLa cells (Greiner et al., 2013). As reported for hederacoside C and liquiritigenin, both the expressions of NF-κB p65 and I-κB and the phosphorylation of these proteins can be inhibited (Akhtar et al., 2019; Kim et al., 2008). Plantamajoside-induced decrease in the expressions of I-κBα and of NF-κB/p65 may be interlinked with the mitogen-activated protein kinase (MAPK) signaling pathway (Wu et al., 2016; Liu & Meng, 2018). Notably, luteolin was simultaneously reported to decrease the miRNA-132 expression, which may constitute a perspective topic for further pharmacological research (Liu & Meng, 2018; Dong et al., 2021).

Experimental methods

Suitable methods are yet to be developed. To date, special assays and gene expression measurements have been reported in research articles.

Pharmacological studies on the immunomodulatory effects on the extracellular matrix

Very little is known about the effects of the plants used in the cough therapy on the extracellular matrix of the upper airways. The extracts from Althaeae radix and Malvae sylvestris flos showed certain in vitro inhibition of the Hyal-1. IC₅₀ was in the order of units of mg/mL (Sendker et al., 2017; Orlando et al., 2015). No data from in vivo animal models are available, and hence the real significance of this effect is somewhat speculative.