INTRODUCTION

The genus Periploca, belonging to family Asclepiadaceae, comprises of about 12 species (Rehman et al., 2003). It is represented in Saudi Arabia by four species: P. aphylla Dcne (Zahran and Younes, 1990), P. somaliensis Browicz (Abdel-Sattar et al., 2009), P. visciformis Vatke (Al-Farhan et al., 2005) and P. Brevicoronata (Goyder and Boulos, 1991). P. aphylla, locally known as Suwwas, is a leafless shrub widely distributed in South Hijaz and Najd regions of Saudi Arabia (Chaudhary and Al-Jowaid, 1999). It is used by Saudi indigenous medicine practitioners as stomachic, laxative and for treatment of swellings and cerebral fever (Kazimierz, 1966; Baquar, 1989), while its latex is applied as a poultice in tumors and swellings (Al-Yahya et al., 1990). Phytochemical studies on different species belonging to genus Periploca resulted in the isolation and structure identification of different classes of secondary metabolites such as pregnane glycosides from P. sepium and P. calophylla (Itokawa et al., 1987; 1988a-c; Xu et al., 1990; Deepak et al., 2012) and cardinolides from P. sepium and P. forrestii (Xu et al., 1990, Li et al., 2010, 2012). In addition, triterpenes, lignanes (Rehman et al., 2003) and flavonoids (Yang-Min et al., 2010) were also reported from genus Periploca.

Biological studies of extracts and isolated compounds from different Periploca species showed to possess cytotoxic immunosuppressive (Feng et al., 2008), antibacterial (Rehman et al., 2003), antioxidant (Iqbal et al., 2012), hepatoprotective and antiprotozoal (Al-Musayeib et al., 2012) activities. However, to the best of our knowledge, no study exists in the literature to validate its medicinal use in gut disorders such as constipation. Knowing that the laxative effect is usually present in the aqueous fractions and that the laxative effect usually co-exists in nature with spasmolytic component present in the non-polar fractions, this study was conducted on the aqueous and n-hexane fractions using both in-vivo and in-vitro studies to validate the medicinal use of plant in gut disorders. .

MATERIAL AND METHODS

Plant material: The aerial parts of P. aphylla were collected in March 2008, from Oyoun village located near Al-Baha city in the south western part of the Kingdom of Saudi Arabia. The plant was identified by Professor Mohammed Yousef, Pharmacognosy Department, College of Pharmacy, King Saud University, Saudi Arabia. A voucher specimen was deposited at the department.

Extract preparation: The air-dried powdered aerial parts (200 g) were exhaustively extracted by ethanol (70%) using Soxhlet apparatus for 8 h. The dried alcoholic extract was then dispensed in 250 mL water and then successively partitioned with several portions of n-hexane (5x350 mL), chloroform (5x350 mL), ethyl acetate (5x350 mL) and n-butanol (5x350 mL). The obtained extracts were separately dehydrated by passing over anhydrous sodium sulfate, evaporated to dryness under reduced pressure, kept in desiccator over anhydrous calcium chloride and saved for further investigation.

The aqueous and n-hexane fraction of the titled plant was screened for its laxative and antidiarrheal effect, while the other extracts were saved for further phytochemical and biological studies.

Drugs: The following reference chemicals were obtained from the sources specified: acetylcholine chloride (Ach), loperamide hydrochloride, carbamoylcholine (carbachol), verapamil hydrochloride, potassium chloride (Sigma Chemical Company, St. Louis, MO, USA) and castor oil (Karachi Chemical Industries, Karachi, Pakistan). Chemicals used for making physiological salt solutions including potassium chloride, calcium chloride, glucose, magnesium chloride, magnesium sulfate, potassium dihydrogen phosphate, sodium bicarbonate, sodium dihydrogen phosphate and sodium chloride were obtained from Merck (Darmstadt, Germany). All chemicals used were of the highest purity grade. Stock solutions of all the chemicals were made in distilled water and the dilutions were made fresh in normal saline on the day of experiment.

Animals: BALB/c mice (weighing 20-25 g ), local breed rabbits (weighing 1-1.5 kg) and guinea pig (400-500 g) of either sex, were housed at the animal house of the Aga Khan University under a controlled environment (23-25°C). The animals were kept in their respective cages with sawdust (changed at every 48 h) and were fasted for 24 h before starting the experiment. In routine, they were given tap water ad libitum and a standard diet consisting of (g kg^-1): flour 380, fiber 380, molasses 12, NaCl 5.8, nutrivet L 2.5, potassium metabisulfate 1.2, vegetable oil 38, fish meal 170 and powdered milk 150. The experiments were performed with the rulings of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (NRC, 1996).

In-vivo experiments
Laxative activity test: Mice fasted for 6 h before the experiment were placed individually in cages lined with clean filter paper. The animals were divided into seven groups (n = 6); the first group acting as the negative control and administered saline (10 mL kg^-1, p.o.), while the next group received CCh (1 mg kg^-1, i.p.) which served as the positive control. The third and fourth groups received orally, 30 and 100 mg kg^-1 of Pa.Aq, respectively. To determine the mechanism underlying its laxative effect, separate sets of mice (group # 5, 6 and 7) were pretreated with atropine (10 mg kg^-1, i.p.) one hour before administration of the extract or CCh. After 18 hours, the feces production (total number of feces and total number of wet feces per group) in all animals was counted and the percentage increase in wet feces relative to that of total fecal output was recorded which was considered as the laxative effect (Khan et al., 2012).

Antidiarrheal activity: The method previously used in our lab was followed with some modifications. Mice (20-25 g) of either sex were fasted for 24 h before the experiment. The animals were housed in individual cages and divided in four equal groups, for each n = 5. The first group received saline along with normal saline (10 mL kg^-1, p.o.), acted negative control. The second and third groups received Pa.Hex 30 and 100 mg kg^-1, respectively. Fourth group received loperamide (10 mg kg^-1), as positive control. Afterwards, the cages were inspected for the presence and absence of typical diarrheal droppings; the absence was noted as a positive result, indicating protection from diarrhea.

In-vitro experiments: The spasmogenic/spasmolytic activities were studied on isolated rabbit jejunum and guinea pig ileum preparations as described previously (Syed Taqvi et al., 2006; Janbaz et al., 2013; Khan et al., 2012). Approximately 2 cm long segments of jejunum or ileum were suspended in tissue baths containing Tyrode’s solution maintained at 37°C and aerated with carbogen (95% O₂ and 5% CO₂). Intestinal responses were recorded isotonically using Bioscience transducers attached to Powerlab Data Acquisition System (AD Instruments, Sydney, Australia) linked to a computer installed with Labchart software (version 6). The tissues were allowed to equilibrate for 30 min prior to addition of any chemical substance. The tissues were stabilized following repeated exposure to 0.3 μM acetylcholine (3-5 times) after washing with the Tyrode’s solution until the sub-maximal responses of uniform amplitude were obtained. The observed modulation of spontaneous rhythmic contractions were used to test spasmogenic or spasmolytic activity in isolated rabbit jejunum preparation, whereas, induction of contraction with test or control drugs above that of the basal tone was used to measure spasmogenic activity in guinea-pig ileum.

Calcium channel blocking activity: To assess whether the spasmolytic activity of the test substances was mediated through calcium channel blockade, high concentration of potassium as, K⁺ (80 mM), was used to depolarize the jejunal preparations (Farre et al., 1991). K⁺ (80 mM) was added to the tissue bath which produced a sustained contraction. Test material and standards were then added in a cumulative fashion to obtain concentration-dependent inhibitory responses. The relaxation of intestinal preparations, pre-contracted with high K⁺, was expressed as percent of the control pre-contraction. To confirm the calcium antagonist activity of test substances, 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. This solution was further replaced with K⁺-rich and Ca⁺⁺-free Tyrode's solution, having the following composition: KCl 50, NaCl 91.04, MgCl₂ 1.05, NaHCO₃ 11.90, NaH₂PO₄ 0.42, glucose 5.55 and EDTA 0.1 mM, with pH of 7.4. Following an incubation period of 30 min, control concentration-response curves of Ca⁺⁺ were obtained. When the control concentration response curves of Ca⁺⁺ were found super-impossible (usually after two cycles), the tissues were pretreated with the test material for 60 min to test the possible calcium channel blocking effect. The concentration response curves of Ca⁺⁺ were reconstructed in the presence of different concentrations of the test material.

Statistical analysis: The data expressed are Mean±standard error of mean (SEM, n = number of experiments) and the median effective concentrations (EC₅₀ values) with 95% confidence intervals (CI). The concentration-response curves (CRCs) were analyzed by non-linear regression while Chi-square-test for antidiarrheal assay. All the graphs, calculations and statistical analysis were performed using GraphPad Prism 4 for windows (GraphPad Software, San Diego, California, USA).