Antidiarrheal, antisecretory, and bronchodilatory activities of Hypericum perforatum

Abstract

This study describes the antidiarrheal, antisecretory, and bronchodilatory activities of Hypericum perforatum Linn. (Hypericaceae), commonly known as St. John’s wort, to justify its traditional use in the hyperactivity of the gastrointestinal and respiratory systems. The crude extract of Hypericum perforatum (Hp.Cr) at a dose of 500 mg/kg caused 20% protection against castor oil-induced diarrhea in mice and 60% at 1000 mg/kg (p < 0.05 vs. saline). Hp.Cr at 300 and 1000 mg/kg reduced the castor oil-induced fluid accumulation in mice to 107.0 ± 3.3 g (p < 0.01) and 84.0 ± 4.2 g (p < 0.001) respectively, whereas in the castor oil-treated group, it was 126.9 ± 3.9 g. When tested against carbachol (CCh)-mediated bronchoconstriction in rats under anesthesia, Hp.Cr dose-dependently (3– 30 mg/kg) suppressed the CCh (1 μmol/kg)-induced increase in the inspiratory pressure. Thus this study rationalizes the Hypericum perforatum usefulness in overactive gut and airways disorders, such as diarrhea and asthma.

Keywords::

• Hypericum perforatum
• St. John’s wort
• in vivo
• antidiarrheal
• antisecretory
• bronchodilatory

Introduction

Hypericum perforatum Linn. (Hypericaceae) is commonly known as St. John’s wort. The genus Hypericum consists of more than 370 species (Baquar, 1989; Nahrstedt & Butterweck, 1997). Among the various species of Hypericum, the plant Hypericum perforatum is well known for therapeutic efficacy. Euryphon, a Greek physician in 288 bc, first described the medicinal value of Hypericum perforatum, and it was prescribed by Hippocrates himself (Mahady et al., 2001). Since the middle of the 19th century, the application of St. John’s wort for mild to moderate depression has become prominent worldwide. Its use in crude form (botanical) for the treatment of mild to moderate depression is a common practice of modern physicians in Germany and other European countries (Miller & Murray, 1998; Muller, 2003).

Hypericum perforatum is used in folk medicine to treat asthma, bronchitis, diarrhea (Nadkarni, 1976; McKenna et al., 2002), excoriations, hemorrhoids, and wounds (Baquar, 1989). It is also considered useful as an anthelmintic, astringent, detersive, diuretic, and emmenagogue. The herb is used to treat burns, eczema, and hysteria (Barnes et al., 2001; Mateo et al., 2002). Its oil is taken orally to treat abdominal spasm, gastritis, gastric ulcers, and inflammatory conditions of the colon (Weiss, 1988). The powdered drug or extracts of the plant are used as an antirheumatic, as well as for supportive treatment of nervous excitement and sleep disturbances (Wyk et al., 2002). It is known to be a useful herbal remedy for the treatment of neurological disorders, such as coxalgia, menopausal neurosis, headache, hypersensitivity, mental ailments, neuralgia, paralysis, spinal convulsions, stiff neck, and tetanus (Ozturk et al., 1996).

Phytochemical studies revealed the presence of multiple chemicals in the plant, such as ascorbic acid, brenzcatechin, cadinine, carotene, carotenoids, catechin, choline, emodinantherol, epicatechin, gurjunene, hyperforin, hypericin, hyperoside, imanin, isohype-ricin, isoquercitrin, limonene, mannitol, myristic acid, pectin, phenol, phlobaphene, phloroglucinol, protohypericin, pseudohypericin, pyrogallol, quercetin, quercitrin, resorcynol, rutin, saponin, tannins (Duke, 1992; Bombardelli & Morazzoni, 1995), amino acids, biflavones, essential oils, flavonols, glycosides, naphthodianthrones, phenylpropanes, proanthocyanidins, and xanthones (Nahrstedt & Butterweck, 1997; Wheatley, 1998).

Hypericum perforatum has been known to possess antidepressant (Harrer et al., 1999; Josey & Tackett, 1999; Woelk, 2000), anxiolytic (Kumar et al., 2000), antioxidant (Tripathi & Pandey, 1999), antibacterial (Keles et al., 2001), antifungal (Khosa & Bhatia, 1982), anti-inflammatory, analgesic (Kumar et al., 2001), antiviral (Serkedjieva et al., 1990), hepatoprotective (Ozturk et al., 1992), sedative (Girzu et al., 1997), memory enhancing (Khalifa, 2001), and nephroprotective (Shibayama et al., 2007) properties.

We have recently reported the antispasmodic and tracheo-relaxant effects of St. John’s wort in isolated tissue preparations, mediated through a combination of Ca²⁺ antagonist and phosphodiesterase (PDE)-inhibitory mechanisms (Gilani et al., 2005a). The present in vivo studies were carried out to validate its effectiveness against the hyperactive state of the gut and airways.

Materials and methods

Chemicals

Atropine sulfate, carbachol (CCh), loperamide, and theophylline were purchased from Sigma Chemical Co., St. Louis, MO, USA. Pentothal sodium (thiopental sodium) and castor oil were respectively obtained from Abbott Laboratories and KCL Pharma, Karachi, Pakistan. All chemicals used were of the analytical grade available.

Animals

Animals used in this study, Sprague-Dawley rats (200–250 g) and Balb-C mice (20–25 g) of either sex, were of local breed, housed at the Animal House of The Aga Khan University, maintained at 23–25°C. Animals were given tap water ad libitum and a standard diet consisting of (g/kg): flour 380, chokar 380, molasses 12, NaCl 5.8, Nutrivet L 2.5, potassium bisulfate 1.2, vegetable oil 38, fish meal 170, and powdered milk 150. Experiments performed complied with the rulings of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (1996) and were approved by the Ethics Committee of The Aga Khan University.

Plant material and preparation of crude extract

The aerial parts (stem and leaves) of Hypericum perforatum were collected from the Northern Areas of Pakistan (Gillyat) in May 2002. The plant was identified with the help of a taxonomist, Dr. M. Ibrar, at the Department of Pharmacy, University of Peshawar, Peshawar, NWFP, Pakistan. A voucher specimen (PUP 012529) was submitted to the Herbarium of the Department of Botany of the same university. The plant material was cleaned, dried, and coarsely ground. The powdered material was extracted with 70% aqueous-ethanol by cold maceration for 3 days with occasional shaking. It was filtered through a muslin cloth and then through a Whatman qualitative grade 1 filter paper (Williamson et al., 1998). This procedure was repeated twice, and the combined filtrate was evaporated on a rotary evaporator under reduced pressure (–760 mmHg) to a thick, semi-solid mass of dark brown color, i.e., crude extract of Hypericum perforatum (Hp.Cr), yielding approximately 18.52% (w/w). Hp.Cr was completely solubilized in saline (0.9%).

Castor oil-induced diarrhea

Mice were fasted for 24 h before the experiment (Das et al., 1999; Gilani et al., 2005b; Borelli et al., 2006). Animals were housed in individual cages and divided into four groups, each containing five mice. The first group received saline (10 mL/kg, p.o.) and served as a negative control. The doses of test extract were selected on a trial basis and two increasing doses were given orally. A group of mice was treated with loperamide ( 10 mg/kg, p.o.), as a positive control. One hour after treatment, each animal received 10 mL/kg of castor oil orally through a feeding needle. Afterward, the cages were inspected for the presence of diarrhea droppings; their absence was noted as a positive result, indicating protection from diarrhea at that time.

Intestinal fluid accumulation

Intestinal fluid accumulation was studied by the enteropooling assay (Izzo et al., 1994; Capasso et al., 2002). Different groups of overnight-fasted mice were treated with increasing doses of extract intraperitoneally through a detachable U-100 insulin syringe with a 25G × 1 in (0.50 × 25 mm) needle, 1 h before the administration of castor oil (10 mL/kg, p.o.). The mice were sacrificed 30 min later by cervical dislocation and the entire intestine was removed and weighed with care, not allowing any intestinal fluid to leak out. The results are expressed as (Pi/Pm) × 1000, where Pi is the weight (g) of the intestine and Pm is the weight of the animal (Gilani et al., 2008a).

Bronchodilatory activity

Rats were anesthetized with sodium thiopental (Pentothal; 80– 100 mg/kg, i.p.), then intubated with a tracheal tube and ventilated with a volume ventilator (Miniature Ideal pump; Bioscience, UK) adjusted to a rate of 70–80 strokes/min to deliver 7–10 mL/kg of room air (Dar & Channa, 1997; Channa et al., 2005). A polyethylene catheter was inserted into the jugular vein for drug administration. Changes in airway resistance (mmHg) were measured by a pressure transducer (MLT-1199) connected to the side arm of a tracheal cannula and recorded by a PowerLab 4/25 with running chart software via a Quad bridge amplifier (ADInstruments, Bella Vista, NSW, Australia). Bronchoconstriction was induced with CCh (1 μmol/kg), which was reversed within 7–10 min. The test drug was given to the animals 5–8 min prior to administration of CCh. The responses are expressed as percent reduction of the CCh-induced bronchospasm (Gilani et al., 2008b).

Acute toxicity test

Animals were divided into groups of five mice each. The test was performed using increasing doses of test extract, given orally, in a 10 mL/kg volume to different groups serving as test groups (Sanmugapriya & Venkataraman, 2006). Another group of mice was administered saline (10 mL/kg, p.o.) as negative control. The mice were allowed food ad libitum during the 24 h test and kept under regular observation for mortality.

Statistical analysis

The data are expressed as mean ± standard error of the mean (SEM) and were analyzed using the GraphPad program (GraphPad, San Diego, CA, USA). The statistical parameter applied was Student’s t-test except in the case of antidiarrheal study, where the χ² test was used. p < 0.05 was noted as significantly different.

Results

Effect on castor oil-induced diarrhea

Hp.Cr exhibited a dose-dependent (500– 1000 mg/kg) protective effect against castor oil-induced diarrhea in mice. The negative control treatment (saline) did not protect the animals from diarrhea. Pretreatment of animals with Hp.Cr produced 20% protection from diarrhea at 500 mg/kg and 60% protection at 1000 mg/kg (p < 0.05 vs. saline group). Loperamide ( 10 mg/kg), a standard antidiarrheal agent, showed complete protection (100%, p < 0.01 vs. saline group) from diarrhea in the positive control group (Table 1).

Table 1.  Effect of the crude extract of Hypericum perforatum (Hp.Cr) on castor oil (C.oil, 10 mL/kg)-induced diarrhea in mice.

	No. of mice/5 with diarrhea	% Protection
Saline (10 mL/kg) + C.oil	5	0
Hp.Cr ( 500 mg/kg) + C.oil	4	20
Hp.Cr ( 1000 mg/kg) + C.oil	2*	60
Loperamide ( 10 mg/kg) + C.oil	0**	100

*p < 0.05, **p < 0.01 compared to saline group, χ2 test.

Effect on intestinal fluid accumulation

When tested against castor oil-induced intestinal fluid accumulation in mice, Hp.Cr exhibited a dose-dependent (300– 1000 mg/kg) antisecretory effect. Intestinal fluid accumulation in the saline treated group was 94.6 ± 3.2 g (mean ± SEM, n = 5), whereas in the castor oil-treated group, it was 126.9 ± 3.9 g (p < 0.001 vs. saline group). Hp.Cr at the doses of 300 and 1000 mg/kg reduced the castor oil-induced fluid accumulation to 107.0 ± 3.3 g (p < 0.01 vs. castor oil group) and 84.0 ± 4.2 g (p < 0.001 vs. castor oil group), respectively (Figure 1). Atropine at the dose of 10 mg/kg decreased the intestinal fluid accumulation to 82.0 ± 3.0 g (p < 0.001 vs. castor oil group) as shown in Figure 1.

Figure 1.  Effect of the crude extract of Hypericum perforatum (Hp.Cr) and atropine on castor oil-stimulated fluid accumulation in small intestine of mice. Results shown are mean ± SEM for five animals in each experimental group. Intestinal fluid accumulation is expressed as Pi/Pm × 1000 (g) where Pi is the weight of the small intestine and Pm is the weight of the mouse. ^#p < 0.001 vs. saline group, **p < 0.01 and ***p < 0.001 vs. castor oil group, Student’s t-test.

Effect on carbachol-induced bronchoconstriction

Hp.Cr at the doses of 3, 10, and 30 mg/kg caused 23.8 ± 2.3, 51.7 ± 4.9, and 72.7 ± 4.1% (n = 4) respective inhibition of the CCh (1 μmol/kg)-evoked increase in inspiratory pressure of anesthetized rats (Figure 2A). Theophylline was used as a positive control, which suppressed the CCh (1 μmol/kg)-induced bronchoconstriction at 1, 3, and 10 mg/kg by 21.5 ± 4.3, 43.4 ± 2.7, and 77.5 ± 6.6% (n = 4) respectively (Figure 2B).

Figure 2.  Dose-dependent effect of (A) crude extract of Hypericum perforatum (Hp.Cr) and (B) theophylline on carbachol (CCh)-mediated bronchoconstriction in anesthetized rats. Values shown are mean ± SEM, n = 4.

Acute toxicity study

The two different groups of mice were given Hp.Cr in the graded doses of 5 and 10 g/kg, respectively, and the animals were observed for mortality 24 h after drug administration. The extract did not cause any mortality up to the dose of 10 g/kg.

Discussion

Since Hypericum perforatum has been used in diarrhea (Nadkarni, 1976; McKenna et al., 2002), its extract was tested for a protective effect against castor oil-induced diarrhea in mice. The induction of diarrhea with castor oil results from the action of ricinoleic acid formed by the hydrolysis of oil (Iwao & Terada, 1962), which produces changes in the transport of electrolytes and water (Gaginella & Phillips, 1975), resulting in the generation of giant contractions of the transverse and distal colon (Croci et al., 1997). Thus, a potential antidiarrheal agent may exhibit its antidiarrheal effect by inhibiting bowel contractions. Hypericum perforatum extract protected the mice against castor oil-induced intestinal fluid secretions, which also contributes to its potential in bowel inflammatory conditions such as gastric ulcers (Weiss, 1988). St. John’s wort was previously observed to suppress irinotecan-induced diarrhea and intestinal lesions through inhibition of proinflammatory cytokines and intestinal epithelial apoptosis (Hu et al., 2006), hence accounting for its gastrointestinal protective profile.

Based on the traditional use of Hypericum perforatum in congestive respiratory disorders (McKenna et al., 2002), it was tested for a possible bronchodilatory effect in anesthetized rats. The extract inhibited CCh-evoked bronchospasm in a dose-dependent manner, like that caused by theophylline, a standard bronchodilator (Rabe et al., 1995), thus justifying its application in asthma and bronchitis. Hypericum perforatum is reported to exhibit calcium channel blocking and PDE-inhibitory effects (Gilani et al., 2005a); hence the present observations (gut and airways inhibitory activities) are in accordance with the expectation, as both Ca²⁺ antagonists and PDE inhibitors are considered effective for such actions (Reynolds et al., 1984; Scratcherd & Grundy, 1984; Sopory et al., 2004; Barnes, 2006).

The flavonoids are well known for their antidiarrheal, antisecretory, and bronchodilatory activities (Di Carlo et al., 1993; Pietta, 1998; Khan & Gilani, 2006; Ghayur et al., 2007) and the presence of these types of compounds, such as quercetin, rutin, catechin, and hyperforin, in St. John’s wort is likely to contribute to its gastrointestinal and respiratory effects, though the likely role of tannins present in the plant cannot be ignored, as the tannins are known to have a beneficial role in diarrhea (Heinrich et al., 1992; Gilani et al., 2006). In acute toxicity testing, the extract was found safe up to the maximum dose (10 g/kg) tested, which is in line with the wide therapeutic use of St. John’s wort.

In conclusion, the present study, by reporting the antidiarrheal, antisecretory, and bronchodilatory effects of Hypericum perforatum, contributes toward evidence-based phytomedicine.

Acknowledgements

The authors are thankful to Dr. Fazal Subhan (Department of Pharmacy, University of Peshawar, Pakistan) for supply of the plant material.

Declaration of interest: This study was supported by funds made available by the Pakistan Science Foundation and Higher Education Commission, Government of Pakistan.