In Vitro Antichemotactic Activity of Chamomilla recutita Hydroethanol Extract

Abstract

In this study, we have investigated the ability of the hydroethanolic extract prepared from the flower heads of Chamomilla recutita. (L.) Rauschert (Asteraceae) to interfere with human leukocyte chemotaxis induced by casein using the in vitro. Boyden system. The results showed a striking dose-related inhibition of the casein-induced human polymorphonuclear leukocyte migration after chamomile extract treatment in comparison with untreated controls, a biological effect never reported before for this ancient plant. Moreover, these effects were similar to those showed by dexamethasone, a model drug. Of particular interest are the results observed after pretreating these cells with morphine, in which the antichemotactic activity could be partially blocked, suggesting that these effects might be opioid receptor mediated. The data herein presented may be useful to clarify the worldwide use of this herbal medicine as an oral infusion, supporting its ethnomedical application as a calming, spasmolytic, and anti-inflammatory agent.

Keywords:

• Casein
• Chamomilla recutita
• chemotaxis
• morphine
• opioid receptors
• polymorphonuclear leukocytes

Introduction

Dried flower heads of Chamomilla recutita (L.) Rauschert (Asteraceae), known simply as chamomile, are used worldwide both internally and externally to treat an extensive list of conditions (Blumenthal, 2000), from skin inflammation (Tubaro et al., 1984; Rekka et al., 1996; Brown & Dattner, 1998; Leung & Foster, 1998) to cancer (Maiche et al., 1991; Lepley et al., 1996; Birt et al., 1997; Hernandez-Ceruelos et al., 2002). Being one of the most well documented medicinal plants, several of its potentially biologically active chemical constituents have been described, some of them isolated from its essential oil (Isaac, 1979; Jakovlev et al., 19791983; Safayhi et al., 1994; Gerritsen et al., 1995; Miller et al., 1996; Rekka et al., 1996; Avallone et al., 2000). For example, its flavonoids have been shown to exert benzodiazepine-like activity (Viola et al., 1995; Avallone et al., 19962000) as well as phosphodiesterase inhibitory action, leading to increased levels of cAMP (Beretz et al., 1978; Picq et al., 1989). Apigenin and chamazulene have been reported to affect the levels of protein upregulation at the transcriptional level (Gerritsen et al., 1995; Rekka et al., 1996; Liang et al., 1999).

Although used in different pharmaceutical preparations according to the need (Tubaro et al., 1984; Mann & Staba, 1986; Maiche et al., 1991; Fidler et al., 1996; Li et al., 1996), chamomile has largely been consumed as a tea or infusion for sedative and anxiolytic purposes (Viola et al., 1995; Cauffield & Forbes, 1999; Larzelere & Wiseman, 2002) as well as a digestive aid to treat gastrointestinal disturbances, particularly in children (Weizman et al., 1993; de la Motte et al., 1997; Mills & Bone, 2000; Madisch et al., 2001).

Because chamomile tea has the properties of relieving digestive conditions and tranquilizing, we have hypothesized that these effects could be mediated through opioid receptors as they are fairly distributed in both systems. To address this question, we have used the in vitro. Boyden chamber system (Boyden, 1962), in which human leukocytes that express opioid receptors (Chuang et al., 1995; Makman et al., 1995; Caldiroli et al., 1999; Sharp, 2003) are stimulated to migrate toward a gradient of casein, a milk-derived protein that has specific binding sites for human leukocytes (Lewis & Van Epps, 1983) and has been shown to contain fragments that behave like opioid receptor ligands, able to exert opioid activity (Brantl et al., 1981; Brantl, 1984). Also, its strong effect on leukocyte chemotaxis has been reported to be mediated through opioid receptors (Makman et al., 1995). As morphine is a classical opioid drug and can influence a variety of biological responses of cells that express opioid receptors (Makman, 1994), its effects under similar experimental conditions have been investigated for comparative purposes.

Materials and Methods

General experimental procedures

Unless otherwise specified, all solid and liquid chemicals used in this work were purchased from Sigma (St. Louis, MO, USA) or Merck (Germany). Dexamethasone sodium phosphate (DEXA), naloxone (Campinas, Brazil), and morphine sulfhate were purchased from Prodome, Narcan (Sao Paulo, Brazil), and Cristália (Sao Paulo, Brazil), respectively.

Plant material

Dried flower heads of Chamomilla recutita (L.) Rauschert (Asteraceae), imported from Egypt and purchased in a free market in Curitiba, Parana state, Brazil, were identified by pharmacognostic analysis, and a voucher specimen has been deposited at the herbarium of the Laboratory of Pharmacognosy, Pharmacy Department, Federal University of Paraná, Brazil.

Extract preparation

Plant material (50 g) was extracted at room temperature in a percolator containing 20% ethanol (200 mL) for 48 h. The resulting solution was filtered, evaporated to dryness in a rotavapor at 37°C, resuspended into Dulbecco's PBS, filter-sterilized (Acrodisc [Ann Arbor, MI, USA]), aliquoted, and kept frozen at − 20°C until needed. Part of the plant material was subjected to hydrodistillation for 2 h in a Clevenger-type apparatus for essential oil extraction, which was dried over anhydrous sodium sulfate and, after filtration, maintained under refrigeration before analysis to identify its components by gas chromatography (HP 6890 [Hewlett Packad, Wilmington, DE, USA]) coupled with mass spectroscopy (GC-MS) according to the methodology described elsewhere (Scalia et al., 1999).

Isolation of human leukocytes

The protocol used in this work for peripheral blood collection has been approved by the Ethical Committee of the Hospital de Clinicas, Universidade Federal do Paraná (CEP-HC N. 066EXT020-11). Peripheral blood samples (5–8 mL) were obtained by venipuncture from healthy volunteers after consent and collected in EDTA-K₃. Buffy coats were obtained after spinning the samples at 800 × g. for 25 min at room temperature and collecting the upper, leukocyte-rich fraction. Contaminating erythrocytes were removed by osmotic lysis with Gey's lysis buffer (solution A: NH₄Cl 654.20 mM, KCl 24.83 mM, Na₂HPO₄ · H₂O 6.53 mM, KH₂PO₄ 0.88 mM, glucose 27.77 mM, phenol red 0.05 g/l; solution B: MgCl₂ · 6H₂O 2.06 mM, MgSO₄ · 7H₂O 0.56 mM, CaCl₂ 3.42 mM; solution C: NaHCO₃ 26.78 mM, all at pH 7.2–7.4; just before use, 4:1:1 parts of solutions A, B, and C were, respectively, added to 14 parts of H₂O, filter-sterilized, and kept at room temperature). After washing twice in PBS (NaH₂PO₄ · 2H₂O 150 mM; Na₂HPO₄ 150 mM; NaCl 154 mM, pH 7.2), the remaining leukocytes were resuspended into PBS supplemented (PBSs.) with glucose (0.1% w/v), BSA (0.25% w/v), CaCl₂ (0.9 mM), and MgCl₂ (0.5 mM), and the cell concentration adjusted to 10⁶/mL. The cell preparations contained more than 95% viable cells as estimated by the Trypan blue exclusion test.

Cytotoxicity of the extract and drugs

Toxic effects of the plant extract, dexamethasone, morphine, and naloxone solutions upon human leukocytes were measured for all concentrations assayed using the Trypan blue exclusion test.

Leukocyte migration

Leukocytes were incubated for 30 min at 37°C with increasing extract concentrations (0.1–1000 µg/mL), washed twice with PBS, and resuspended into PBSs. Thereafter, migration of the treated cells toward casein was measured using a modified Boyden technique (Boyden, 1962). Briefly, the upper chamber was separated from the lower one by a 5-µm pore size, PVP-free polycarbonate filter (NeuroProbe [Gaithersburg, MD, USA]) and loaded with 10⁵ human leukocytes, which were stimulated to migrate toward 1% (w/v) casein gradient placed into the lower chamber for 90 min at 37°C. Polymorphonuclear cell (PMN) migration was expressed as a percentage of the control, That is, the number of cells that had not been treated with any plant extracts migrating toward chemoattractant under similar conditions. In some experiments, cells were pre treated for 30 and 10 min at room temperature with dexamethasone (DEXA; 10⁻⁵ M), morphine or naloxone (both at 10⁻⁷ M), respectively, before incubation with the chamomile extract. In others, cells were first treated with the plant extracts followed by the drugs.

Statistical evaluation

The results are expressed as the mean percentage±standard error of the mean (SEM). Statistical significance was calculated by the t.-test, and differences were considered significant when p ≤ 0.05.

Results

Primary screening tests done with the chamomile extract and drugs used in this study showed no signs of toxicity to the human leukocytes, which were bright and of round shape and their viability always greater than 98%, before and after the chemotaxis experiments. The phytochemical screening of the chamomile extract revealed, as expected, the presence of flavonoids and tannins, and the GC-MS chromatographic profile of the essential oil (not shown) was similar to that recently reported by others (Scalia et al., 1999), supporting the identity of the purchased herb.

The effects of exposing human leukocytes from peripheral blood to increasing concentrations of chamomile extract before inducing them to migrate toward a casein gradient is shown in Figure 1 and resulted in an increased dose-related number of cells retained in the upper chamber compartment in comparison with the untreated control up to 10 µg/mL, at which concentration it reached maximum significance, with only 65.8±5.3% (n = 15; p ≤ 0.005) of the cells recovered from the lower chamber. For DEXA, which has the ability to inhibit the migration properties of leukocytes in vitro. (Lomas et al., 1991; Zentay et al., 1999) and has been considered as a model for this purpose, the value found using similar conditions was 69.1±6.2% (n = 17; p ≤ 0.005, when compared with the control group).

Figure 1 Effects of chamomile extract on the casein-induced chemotactic activity of human leukocytes. Peripheral blood leukocytes obtained from healthy volunteers pretreated for 30 min at 37°C with DEXA (10⁻⁵ M) or chamomile extract at indicated concentrations were induced to migrate toward 1% casein gradient for 90 min at 37°C in a Boyden chamber in which the compartments were separated by a 5-µm pore size polycarbonate filter. Each column represents the mean %±SEM of total cells recovered from the lower compartment in relation to untreated cells, normalized at 100% from at least three individual experiments (*p < 0.05; **p < 0.025; ***p < 0.0005).

Figure 2 depicts the results of pretreating human leukocytes with morphine or naloxone before chemotaxis toward casein gradient. Their exposure to these drugs resulted in the migration of 82.6±2.3% (n = 9; p < 0.0005) and 83.9±2.5% (n = 8; p < 0.0005) of PMNs, respectively, and are in agreement with the inhibitory effect shown by these molecules using casein or even other chemotactic agents such as N.-formyl-methionyl-leucyl-phenylalanine peptide or activated serum (Marcoli et al., 1988; Pasotti et al., 1992). Pretreatment with naloxone did not revert the inhibitory effects of morphine as expected. On the contrary, a significant enhancement in the number of leukocytes retained in the upper compartment was found, with only 67.7±5.6% (n = 4; p < 0.025) of PMNs migrating toward casein.

Figure 2 Effects of opioids on casein-induced chemotaxis of human leukocytes. Peripheral blood leukocytes obtained from healthy volunteers pretreated for 10 min at 37°C with morphine and/or naloxone (10⁻⁷ M) were induced to migrate toward a 1% casein gradient for 90 min at 37°C in a Boyden chamber in which the compartments were separated by a 5-µm pore size polycarbonate filter. Each column represents the mean %±SEM of total cells recovered from the lower compartment in relation to untreated cells, normalized at 100% from at least four individual experiments (**p < 0.025; ***p < 0.0005).

To further examine the possible involvement of opioid receptors in the mechanisms of cell migration inhibition mediated by chamomile extract, human leukocytes were treated with morphine (Fig 3) or naloxone (Fig 4) before and after exposure to the plant extracts followed by their exposure to casein. While pretreatments with morphine or naloxone followed by treatment with chamomile extracts have shown recovery of 78.4±6.2% (n = 4; p < 0.025) and 83.2±5.4% (n = 4; p < 0.025) of PMNs, respectively, the results of treating cells with chamomile extracts before the opioid drugs were 71.0±6.8% (n = 4; p < 0.05) and 73.4±9.6% (n = 4; p < 0.05) of PMNs, respectively.

Figure 3 Effects of chamomile extract and morphine on casein-induced human leukocyte chemotaxis. Peripheral blood leukocytes obtained from healthy volunteers pretreated with chamomile extract (10 µ/mL) or morphine (10⁻⁷ M) followed by treatment with morphine or plant extract were induced to migrate toward a 1% casein gradient for 90 min at 37°C in a Boyden chamber in which the compartments were separated by a 5-µm pore size polycarbonate filter. Each column represents the mean %±SEM of total cells recovered from the lower compartment in relation to untreated cells, normalized at 100% from at least four individual experiments (**p < 0.025).

Figure 4 Effects of chamomile extract and naloxone on casein-induced chemotaxis of human leukocytes. Peripheral blood leukocytes obtained from healthy volunteers pretreated with chamomile extract (10 µg/mL) or naloxone (10⁻⁷ M) followed by treatment with naloxone or plant extract were induced to migrate toward a 1% casein gradient for 90 min at 37°C in a Boyden chamber in which the compartments were separated by a 5-µm pore size polycarbonate filter. Each column represents the mean %±SEM of total cells recovered from the lower compartment in relation to untreated cells, normalized at 100% from four individual experiments (*p < 0.05).

Discussion and Conclusions

Although the flower heads of chamomile have been used therapeutically for several conditions, its extract has been considered to have two specific fields of action: the nervous system (as a calming treatment) and the gastrointestinal tract (decreasing irritation and as carminative and spasmolytic) (Leung & Foster, 1998; Mills & Bone, 2000). Because both systems involve opioid receptors, and to gain more insight into the mechanisms responsible for the efficacy of chamomile infusion in the treatment of disturbances related to them, we have stimulated human leukocytes that recently have shown expression of the various mRNAs encoding the same opioid receptors originally identified in neuronal tissues of central and peripheral origin (Sharp, 2003) to migrate toward casein in a Boyden chamber system, which has been extensively used as a model for investigating specific effects of plant extracts upon leukocyte locomotion (Muller et al., 1999; Hofbauer et al., 20002001; Shen et al., 2001).

Casein was used as chemoattractant because it and its peptides have long been known to interact with specific cell surface receptors expressed on human leukocytes, particularly PMNs, the first line of the body's defense against invaders, inducing their migration. Also, when orally administered to humans (Charlin et al., 1992; Madrid et al., 1996) or in animal models (Defilippi et al., 1995; Defilippi & Gomez, 1995), casein has been shown to decrease the amplitude and frequency of small intestinal contractions and to induce mucus secretion (Claustre et al., 2002), which were suppressed by pretreatment with naloxone, suggesting the involvement of opioid receptors.

Our results showed a significant inhibitory effect of chamomile extract upon casein-induced leukocyte chemotaxis, which was close to the significant effects caused by DEXA, an anti-inflammatory drug commonly used in this system as a negative regulator of leukocyte chemotaxis (Lomas et al., 1991; Zentay et al., 1999). Moreover, the independent pretreatment of these cells with morphine or naloxone before their exposure to the plant extract reduced this activity. Furthermore, they have recapitulated the effects shown by the opioid drugs themselves, suggesting the usage of common, although specific for each drug, sites of interaction by components present in the plant extract. This hypothesis is also supported by the fact that naloxone could not revert the effects of morphine in the system tested. On the contrary, an additive inhibitory effect showed by morphine was observed when the cells were pretreated with naloxone, which was similar to that caused by the extract alone. Thus, the inhibitory effect on leukocyte chemotaxis induced by the chamomile extract observed in these series of experiments may be explained by its interaction with different types or subtypes of opioid receptors expressed on human leukocytes that also interact with morphine and naloxone.

Pharmacological evidence that demonstrates the presence of classical opioid receptors as well as nonclassical opioid-like receptors on cells participating in the host defense and immunity has recently been reported (McCarthy et al., 2001; Kaczor & Matosiuk, 2002a2002b). Morphine and naloxone are drugs that interact with opioid receptor subtypes with different binding affinities and for which specific receptors on human leukocytes have been described (Simpkins et al., 1986; Makman, 1994; Makman et al., 1995; Sharp, 2003).

Inhibition of PMN and monocyte chemotaxis to both complement-derived chemotactic factors (Liu et al., 1992) or to the chemokines MIP-1α. (macrophage inhibitory protein 1-α.), RANTES (regulated upon activation, normal T-cell expressed and secreted), MCP-1 (monocytes chemoattractant protein 1), or IL-8 (interleukin 8) (Grimm et al., 1998b) after morphine and other opioids pretreatment has also been reported. In contrast with our data, these effects could be reversed by naloxone. This disparity of results is not immediately obvious but may be due to the capacity of naloxone to interact with other receptors distinct from the classical opioid receptors that bind morphine present on these challenged cells or to endogenous opioid receptors production and release after activation, as described for peripheral mononuclear cells after stimulation (Buratti et al., 1998). On the other hand, they may be due to mechanisms related to naloxone stereospecificity (Falke et al., 1985) or to more complex interactions, including a crosstalk between opioids and chemokine receptors that would lead to their desensitization as already suggested by others (Grimm et al., 1998a; Szabo & Rogers, 2001; Szabo et al., 2002). In this context, it is of value to note that the mechanism of casein-induced chemotaxis of human (Siddiqui et al., 1999) or animal (Mori et al., 1994) leukocytes seems to be IL-8–mediated, a chemokine that has strong ability for recruiting PMNs to the injury sites (Baggiolini, 1998). The fact that chamomile extract could prevent this stimulatory activity suggests, at least in part, the involvement of chemokines and their receptors in the process.

While the mechanisms are not understood yet, the current investigation has shown that exposure of human leukocytes to chamomile extract is associated with impairment of leukocyte functions, possibly due to its interference on opioid receptor expression on these cells. The data herein presented are in accordance with some reports suggesting that phagocytes from morphine and heroin abusers are defective in their casein-induced migration compared with normal individuals (Perez-Castrillon et al., 1992; Mazzone et al., 1996). They are also consistent with the recent report showing that chamomile extract was able to inhibit both the development of morphine dependence and expression of abstinence syndrome in an animal model (Gomaa et al., 2003), contributing to broaden the spectrum of chamomile effects.

Although the influence of chamomile and its compounds has been extensively investigated in inflammation processes, it has not come to our attention that efforts have been made to investigate its influence on the chemotactic activity of human leukocytes, one of the most important steps of the body's inflammatory response. This is surprising, given the broad pharmacological properties attributed to chamomile.

Our findings are of particular relevance in clinical practice as at least two attractive hypotheses that could explain the popular worldwide usage of chamomile infusions for combating a vast list of disturbances can be drawn: the first would be its effect as a sedative and anxiolytic medicine as well as a digestive aid to treat gastrointestinal discomfort as a consequence of its interaction with opioid receptors. The second would be related to its general application for combating inflammatory processes by inhibiting the exacerbated leukocyte migration to the injury sites, alleviating their symptoms and coping with wound healing.

Additional analysis is needed to clarify whether one or more of the known chamomile compounds, particularly the flavonoids, are responsible for the inhibitory migration activity revealed in this work or whether this effect is the result of the interaction of several compounds present in the extract.