Gut modulatory effects of Daphne oleoides are mediated through cholinergic and Ca⁺⁺ antagonist mechanisms

Abstract

Context: The present study describes the spasmogenic and spasmolytic activities of Daphne oleoides Schreb. (Thymelaeaceae), exploring the possible underlying pharmacological mechanisms.

Aim: Pharmacological investigation of Daphne oleoides to provide evidence for its therapeutic application in gastrointestinal motility disorders.

Materials and methods: Methanol crude extract of Daphne oleoides (Do.Cr) was studied in gastrointestinal isolated tissues.

Results: In spontaneously contracting rabbit jejunum preparations, Do.Cr at 0.3–3.0 mg/mL caused moderate stimulation, followed by relaxant effect at the next higher concentrations (5.0–10 mg/mL). In presence of atropine, spasmogenic effect was blocked and the relaxation was emerged, suggesting that the spasmogenic effect of Daphne oleoides is mediated through activation of muscarinic receptors. When tested against the high K⁺ (80 mM)-induced contractions, Do.Cr (0.3–5.0 mg/mL), like verapamil, inhibited the induced contractions, suggesting Ca⁺⁺ channel blockade (CCB) effect. The CCB effect was further confirmed when pre-treatment of the tissue with Do.Cr shifted the Ca⁺⁺ concentration-response curves to the right, similar to that caused by verapamil.

Discussion and conclusion: These results indicate that Daphne oleoides exhibits gut excitatory and inhibitory effects, occurred via cholinergic and Ca⁺⁺ antagonistic pathways, respectively.

Keywords::

• Daphne oleoides
• spasmogenic
• cholinomimetic
• spasmolytic
• calcium channel blocker

Introduction

The genus Daphne (Thymelaeaceae) consists of 70 species found in Europe, Mediterranean region, temperate and subtropical Asia, Malaysia, Philippine Islands, North Africa, and Australia (Nasir & Ali, 1971). It is represented by three species in Pakistan, i.e., Daphne oleoides Schreb., Daphne papyracea Wall., and Daphne retusa Hemsl. (Zhuang et al., 1983). Plants of the genus Daphne are widely used in traditional medicine as an abortificient (Ronlan & Wickberk, 1970). A tincture named “Mezereum” prepared from the ethanol extract of the bark of Daphne mezereum is useful against various skin diseases and neuralgia (Wangner et al., 1987). The plant also showed stimulating effect in vitro on human and gramcycles and the tumour promoting potential which may be induced by the known diterpene esters (Bernhard et al., 1990). Daphne mezereum has found to contain antileukemic diterpenoid (Kupchan & Baxter, 1975). The root of Daphne odora is called “Ruixanggen” in China. It is used in treatment of snake bite and neuralgic pain (Watt, 1963). The methanol extract of Daphne papyracea possesses hypotensive and tranquillizing properties, and flowers of Daphne genkwa have been used as diuretic and in cancer therapy for centuries in Chinese folklore (Sugi & Nagashio, 1977). The same extract also exhibited in vivo inhibitory activity against P-338 lymphocytic leukaemia in mice and “genkwadaphnin” isolated from the same source showed antileukemic activity (Kasai et al., 1981). Daphne oleoides Schreb. (Thymelaeaceae) is a small multibranched shrub found in the Western Himalayas, from Garhwal Westward to Murree, occurring at an altitude of 3000 to 9000 feet, locally known as “Laighonai.” It is known to contain daphneticin-4″-O-α-d-glucopyranoside, daphnetin-8-O-β-d-glucopyranoside, 4-ethoxybenzoic acid, 4-hydroxybenzoic acid, grantioidin, phenolic acids, terpenoids, and undecyl 2,3-dimethoxycinnamate (Nisar-ullah et al., 1999). Daphne oleoides has been used in various traditional healing systems of the world. Its roots and aerial parts have been used in Turkish folk medicine for the treatment of lumbago fever as well as in rheumatic pain (Yesilada et al., 1995). Various extracts and fractions obtained from whole plant of Daphne oleoides have been reported to show potent inhibitory activity against macrophage-derived cytokines, interleukin 1α (IL-1α), interleukin 1β (IL-1β), and tumor necrosis factor (TNF-α; Yesilada et al., 1997). Root of this plant is purgative and the bark and leaves are given in cutaneous affections (Watt, 1972). Infusion of leaves is also used in gonorrhea and applied to abscesses (Baquar, 1989). Daphne oleoides has also been used against malaria and rheumatism in folk medicinal usage of plants in Turkey (Tabata et al., 1993; Fujita et al., 1995). In this investigation, we provide evidence that Daphne oleoides exhibit gut stimulatory and relaxant effects, mediated through muscarinic receptors stimulation and Ca⁺⁺ antagonist mechanisms, respectively.

Materials and methods

Plant material and preparation of extract

The whole plant of Daphne oleoides was collected from Banda Piran, Siran valley, 2900 m above the sea level, District Mansehra, October 2007. It was identified by Prof. Dr. Manzoor Ahmad, plant Taxonomist, Department of Botany, Government Degree College, Abbotabad, NWFP, Pakistan, where voucher specimen (D-0016) has been deposited in herbarium. Plant material was shade dried and ground to powder (1 kg). The extraction was made by using cold percolation method by extracting with methanol (2.5 L) at room temperature for seven days. The extraction was repeated thrice. The combined extract was evaporated to obtain greenish mass, i.e., crude extract of Daphne oleoides (Do.Cr), yielding ~ 3%. Do.Cr was solubilized in distilled water.

Chemicals

Acetylcholine perchlorate (ACh), atropine sulphate, potassium chloride, and verapamil hydrochloride were purchased from Sigma Chemicals Co, St Louis, MO. Chemicals used for making physiological salt solutions were potassium chloride (Sigma Chemicals Co, St Louis, MO), calcium chloride, glucose, magnesium chloride, sodium bicarbonate, sodium dihydorgen phosphate (Merck, Darmstadt, Germany), and sodium chloride from BDH Laboratory supplies, Poole, England. The chemicals used in phytochemical analysis include acetic anhydride, aluminum chloride (Sigma Chemical Co, St Louis, MO), chloroform, hydrochloric acid, methanol, and petroleum ether (BDH Laboratory supplies, Poole, England). All the chemicals used were of analytical grade available.

Animals

Adult rabbits (1.5–2.0 kg) of local breed and either sex were housed at the Animal House of the Aga Khan University, maintained at 23–25°C and were given a standard diet and tap water. Animals had free access to water, but food was withdrawn 24 h prior to experiment. Rabbits were sacrificed by blow on back of the head. Experiments performed complied with the rulings of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (1996), approved by the Ethical Committee of the Aga Khan University.

Phytochemical screening

Preliminary analysis of the plant extract for various phytochemical classes was carried out following the reported methods (Khan & Gilani, 2009). Plant material treated with petroleum ether and subsequently extracted with chloroform was noted for green to pink or pink to purple color after reaction with acetic anhydride and hydrochloric acid in succession to detect sterols and terpenes, respectively. Appearance of yellow color with aluminum chloride reagent detects flavonoids. The observation of yellow florescence under UV light on filter paper impregnated with the vapors from boiling extract indicates the presence of coumarins.

Rabbit jejunum

The jejunum was dissected out by opening the rabbit abdomen, immersed in Tyrode’s solution and cleaned of mesenteries (Gilani et al., 2009). Each segment of about 2-cm length was suspended in a 10 mL tissue bath containing Tyrode’s solution, maintained at 37°C and aerated with a mixture of 95% oxygen and 5% carbon dioxide (carbogen). The composition of the Tyrode’s solution in mM was KCl 2.68, NaCl 136.9, MgCl₂ 1.05, NaHCO₃ 11.90, NaH₂PO₄ 0.42, CaCl₂ 1.8, and glucose 5.55. Intestinal responses were recorded isotonically using Bioscience transducers and a Harvard oscillograph. Each tissue was allowed to equilibrate for at least 30 min before the addition of any drug and then stabilized with a sub-maximal concentration of ACh (0.3 µM) with a 3-min interval until constant responses were recorded. The contractile effect of the test material was assessed as percent of the maximum effect (Gilani et al., 2005a) produced by the control drug, ACh (1 µM).

Determination of Ca⁺⁺ antagonist effect

For the elucidation of Ca⁺⁺ channel blockade (CCB) activity, high K⁺ (80 mM) was used to depolarize the preparations as described by Farre et al. (1991). K⁺ was added to the tissue bath, which produced a sustained contraction. Test materials were then added in a cumulative fashion to obtain concentration-dependent inhibitory responses (Van Rossum, 1963). To confirm the Ca⁺⁺ antagonist effect of the test substance, the tissue was allowed to stabilize in normal Tyrode’s solution, which was then replaced with Ca⁺⁺-free Tyrode’s solution containing EDTA (0.1 mM) for 30 min in order to remove Ca⁺⁺ from the tissues (Gilani et al., 2005b). This solution was further replaced with K⁺-rich and Ca⁺⁺-free Tyrode’s solution of the following composition (mM): KCl 50, NaCl 91.04, MgCl₂ 1.05, NaHCO₃ 11.90, NaH₂PO₄ 0.42, glucose 5.55, and EDTA 0.1. Following an incubation period of 30 min, control concentration-response curves (CRCs) of Ca⁺⁺ were obtained. When the control Ca⁺⁺ CRCs were found super-imposable (usually after two cycles), the tissue was pretreated with the plant extract for 60 min to test the possible CCB effect. The CRCs of Ca⁺⁺ were constructed in the presence of different concentrations of the test material.

Statistical analysis

Data expressed as mean ± standard error of mean (SEM, n = number of experiments) and the median effective concentrations (EC₅₀ values) with 95% confidence intervals (CI). CRCs were analyzed by non-linear regression using GraphPad program (GraphPAD, San Diego, CA).

Results

Phytochemical analysis

Do.Cr was tested positive for the presence of flavonoids, coumarins, sterols, and terpenes.

Effect on spontaneous contractions

Do.Cr at lower concentrations (0.3–3.0 mg/mL) caused contractile effect on spontaneous contractions of rabbit jejunum. The efficacy of the stimulant effect was 13.6 ± 4.5, 32.3 ± 4.3, and 54.3 ± 5.3% (mean ± SEM; n = 3) at 0.3, 1.0, and 3.0 mg/mL, respectively, compared to ACh maximum contraction. The next higher concentrations (5.0–10 mg/mL) did not cause further increase in contraction, instead caused relaxation of spontaneous contractions (Figure 1). The spasmogenic effect was abolished in the presence of atropine (0.3 µM) and the spasmolytic effect was observed with EC₅₀ value of 5.1 mg/mL (4.1–7.0, 95% CI, n = 3) as shown in Figure 1.

Figure 1.  Concentration-response curves of the crude extract of Daphne oleoides (Do.Cr) on spontaneous contractions of isolated rabbit jejunum preparations in the absence and presence of atropine (0.3 µM). The spasmogenic responses are expressed as percent of the acetylcholine maximum (ACh Max.). Values shown are mean ± SEM, n = 3.

Effect on high K⁺-induced contractions

When tested against high K⁺ (80 mM)-induced contractions, Do.Cr caused relaxation (Figure 2) with EC₅₀ value of 2.6 mg/mL (1.5–4.4, n = 3). Verapamil, used as positive control, inhibited the K⁺ (80 mM)-induced contractions, with EC₅₀ values of 0.15 µM (0.1–0.22, n = 3) as shown in Figure 2.

Figure 2.  Concentration-dependent inhibitory effect of Do.Cr and verapamil against high K⁺-induced contractions in isolated rabbit jejunum preparations. Values shown are mean ± SEM, n = 3.

Effect on Ca⁺⁺-curves

Do.Cr concentration-dependently (3.0–5.0 mg/mL, n = 3) shifted the Ca⁺⁺ CRCs to the right (Figure 3A), like that caused by verapamil at 0.3–1.0 µM (n = 4), as shown in Figure 3B.

Figure 3.  Concentration-response curves of Ca⁺⁺ in the absence and presence of different concentrations of (A) Do.Cr and (B) verapamil in isolated rabbit jejunum preparations. Values shown are mean ± SEM, n = 3–4.

Discussion

In spontaneously contracting rabbit jejunum tissues, Daphne oleoides initially produced excitatory effect, like that caused by ACh. Pretreatment of the tissues with atropine, a muscarinic receptor antagonist (Delmendo et al., 1989), abolished the stimulatory effect of plant extract, which indicates that the Daphne oleoides causes gut stimulation via cholinergic pathway. ACh is a neurotransmitter released by the parasympathetic nervous system, mediates its action in gut by activation of M₃ receptor subtypes and hence plays an important physiological role to regulate the peristaltic movements of the gut (Brown & Taylor, 1996; Ghayur & Gilani, 2005a). The observed spasmogenic effect of Daphne oleoides explains its medicinal use as laxative agent in the hypo-motility disorder of the gut, i.e., constipation. The contractile effect was followed by relaxation at next higher concentrations of the extract, indicating the co-existence of spasmolytic constituent(s). The combination of gut inhibitory component(s) with stimulatory effect in Daphne oleoides is probably meant by nature not to allow the spasmogenic effect going beyond the therapeutic limits, where it could have caused abdominal cramps, as is the case with chemical drugs used in constipation, particularly at high doses (Ghayur & Gilani, 2005b).

In earlier studies, we observed that the antispasmodic effect of medicinal plants is usually mediated through blockade of Ca⁺⁺ channels (Gilani et al., 2005c,d, 2007) To elucidate whether the spasmolytic effect of Daphne oleoides is also mediated via similar mechanism, its extract was tested on high K⁺-induced contractions. High K⁺ (> 30 mM) is known to cause smooth muscle contractions through opening of voltage-dependent L-type Ca⁺⁺ channels, thus allowing influx of extracellular Ca⁺⁺ causing a contractile effect (Bolton, 1979; Pietrobon & Hess, 1990; Karaki et al., 1997) and the substance causing inhibition of high K⁺-induced contraction is considered as inhibitor of Ca⁺⁺ influx (Godfraind et al., 1986; Okmura et al., 1993). Do.Cr, like the verapamil, a standard Ca⁺⁺ antagonist (Fleckenstein, 1977), relaxed the high K⁺-induced contractions, indicating its CCB action. The Ca⁺⁺ antagonist effect of Daphne oleoides was further confirmed when it shifted the Ca⁺⁺ CRCs to the right, like that caused by verapamil, used as positive control. Ca⁺⁺ channel blockers have been shown to be beneficial in the gut disorders, resulting from the hyperactivity, such as abdominal cramps and diarrhoea (Pasricha, 2006). Hence, possessing CCB effect makes the Daphne oleoides a useful therapeutic herbal candidate for the remedy of such conditions.

Phytochemical analysis of the Daphne oleoides extract revealed the presence of flavonoids, coumarins, sterols, and terpenes. The observed cholinergic and CCB effects of the extract may be due to the presence of terpenes and flavonoids, as such phytochemicals have been known to exhibit cholinomimetic and Ca⁺⁺ antagonist activities, respectively (Perry, 1996; Revuelta et al., 1997; Burns et al., 2002).

Conclusions

This study indicates that Daphne oleoides possess gut modulatory (stimulatory and inhibitory) effects, mediated through cholinergic and Ca⁺⁺ antagonist mechanisms, respectively.

Acknowledgement

This study was supported by funds made available by the Higher Education Commission of Pakistan under the Scheme of Distinguished National Professor Research Allowance.

Declaration of interest

The authors report no declarations of interest.