Rationalization of Traditional Uses of Berberis lycium in Gastrointestinal Disorders

literature survey, selection, collection and extraction of ABSTRACT Aims : Berberis lycium (Sumbal) is abundantly available in the northern areas of Pakistan and extensively used in local practice for the treatment of several human diseases. The objective of this study was to explore pharmacological basis for its use in gastrointestinal disorders. Materials Methods: Crude aqueous (Bl.Aq) and methanolic (Bl.Meth) extracts of B. lycium were studied on isolated gut preparations of rabbit (jejunum) and guinea pig (ileum) by using in-vitro techniques. Tissues were mounted in tissue organ baths assembly containing physiological salt (Tyrode's) solution, maintained at 37ºC and aerated with carbogen, to assess the spasmogenic and spasmolytic effect and to find out the tissue with atropine ( 0.1 µM ) partially suppressed the contractile effect. Bl.Aq and Bl.Meth caused complete inhibition of high K + ( 80 mM )–induced contraction at 0.3 mg/mL and 0.1 mg/mL respectively and also produced a dose-dependent ( 0.01-0.03 mg/mL ) rightward shift in the Ca ++ concentration-response curve, similar to verapamil. When tested in bolus protocol on isolated guinea pig ileum, Bl.Aq and Bl.Meth caused a dose-dependent spasmogenic effect at 0.01-0.1 mg/mL. Pretreatment of tissue with atropine ( 0.1 µM ) partially suppress the contractile effect. Conclusions: Results indicate that spasmogenic effect was partially mediated through cholinergic activity and spasmolytic effect was mediated through calcium channel blocking activity (CCB), explain its traditional uses in diarrhea, intestinal cramps and other gastrointestinal intestinal disorders.

Despite the wide medicinal uses of B. lycium, no data is available with respect to its effectiveness in gut motility effect. The present study on the aqueous and methnolic extract of B. lycium roots was undertaken to rationalize these traditional uses and to explore mechanistic basis for these medicinal uses.

Plant Material and Preparation of Crude Extract
Fresh plant root of B. lycium was collected from Gilgit (Shikyote) northern area of Pakistan and was then identified by an expert taxonomist of department of biology, University of Sargodha, Pakistan. Plant was shade dried for one week, thoroughly cleaned and coarsely powered by using electric grinder. The aqueous and methanolic extracts were prepared by cold maceration method. Approximately 1Kg coarsely powdered plant material was soaked in distilled water and methanol separately at room temperature for 7days with occasional shaking [21]. Both were filtered through a double layered muslin cloth and subsequently through filter paper. The collected filtrates were concentrated in rotary evaporator separately at 40ºC under reduced pressure (-760mmHg) to a thick, semi-solid mass, they were transferred to Petri-dishs and placed at room temperature to get rid of remaining solvents. The aqueous and methanolic extracts obtained with approximated yield of 3.85% and4.05% respectively were transfer to glass bottles and stored to refrigerator (-4ºC) until used. The stock solutions and their dilutions were made fresh in distilled water at the day of experiment for purpose of evaluating pharmacological activity.

Preliminary Phytochemical Analysis
Qualitative phytochemical analysis of Bl.Aq and Bl.Meth was done for the presence of Alkaloids, Anthraquinones, Coumarins, Saponins, and tannins as described by Tona et al., 1998.

Drugs and Reagents
All the chemicals used in the experiments were of highest purity and research grade and were obtained from the sources specified: acetylcholine chloride, atropine sulfate, verapamil and potassium chloride from sigma chemicals co.

Animals
Rabbits (1.0-1.5kg) and guinea-pigs (500-600g) of local breed and either sex used for experimental work were housed under controlled environmental condition (23-25ºC) at the animal house of University of Sargodha, Sargodha. Animals were given standard diet and tap water. Constituents of diet are shown as following (

Isolated Tissue Experiments
In-vitro experiments were performed according to the protocols as previously followed by Chaudhary et al., 2012; Abdur Rahman et al., 2012.

Rabbit jejunum
Plant extracts were screened for their spasmogenic and spasmolytic activity on isolated tissue of rabbit jejunum. Segments of approximately 2cm length were suspended in 10mL tissue organ bath containing Tyrode's solution, having the following composition in mM: KCl 2.68; NaCl136.9, MgCl 2 1.05, NaHCO 3 11.90, NaH 2 PO 4 0.42, CaCl 2 1.8 and glucose 5.55, aerated with Carbogen (95% O 2 and 5% CO 2 ) at 37°C. Each tissue was given 1g pre-tension and allowed to equilibrate for at least 30 min. and stabilized with the repeated exposure to 0.3μM acetylcholine (3-5 times) and subsequent washing with the Tyrode's solution until the sub-maximal responses were obtained. The dose-response curves of acetylcholine were constructed before the addition of test materials. Maximum response of the tissue was considered to have been achieved when the next higher concentrations of the agonist failed to produce a further increase in response [27]. The contractile effect of the test materials was assessed as the percent of the maximum effect produced by the control drug, acetylcholine (3µM). Intestinal responses were obtained isotonically using BioScience transducers and Powerlab data acquisition system (AD Instruments, Sydney, Australia) attached to a computer installed with labchart software (version 6).

+2 antagonist effect
To assess whether the relaxant effect of the crude extracts was through calcium channel blocking activity, K + was used to depolarize the preparations as described by Farre et al., 1991. K + (80 mM) was added to induce the sustained contraction. Crude extract was then added to the tissue bath in a cumulative fashion to obtain dose-dependent inhibitory responses [29]. The relaxation of intestinal preparations, pre-contracted with K + (80mM) was expressed as percent of the control response mediated by K + . To confirm the calcium antagonist activity of crud extract, 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 calcium from the tissues. This solution was further replaced with K + -rich and Ca ++ -free Tyrode's solution, having the following composition in mM: KCl 50, NaCl 91.04, MgCl 2 1.05, NaHCO 3 11.90, NaH 2 PO 4 0.42, glucose 5.55 and EDTA 0.1. Following an incubation period of 30 min, control concentration-response curves of Ca ++ were constructed. When the control CRCs of Ca ++ were found super-imposable (usually after two cycles), the tissue was pretreated with the crude extract for 50 min to test the possible calcium channel blocking effect. The CRCs of Ca ++ were reconstructed in the presence of different concentrations of the test material, verapamil used as a positive control.

Guinea-pig ileum
Since isolated guinea pig ileum behaves as a quiescent preparation and is considered more suitable for spasmogenic activity [30]. The ileum was dissected out and segments of approximately 2cm length were suspended individually in 10mL tissue organ bath, filled with Tyrode's solution; having composition as described earlier and was aerated with Carbogen at 37ºC. A preload of 1 g was applied to each tissue and kept constant throughout the experiment. Following an equilibration period of 30 min, isotonic contractions to ACh (0.3 µM) were repeated to stabilize the preparation. Stimulant effect of the extract was determined on the resting baseline of the tissue and was assessed as percent of the maximum effect produced by the control drug, ACh (1 µM).

Statistical Analysis
All the data are expressed as mean ± standard error of the mean (S.E.M., n = number of experiments) and the median effective concentrations (EC 50 values) are given with 95% confidence intervals (CI). The statistical parameter applied is the Student's t-test with p< 0.05 considered as significantly. All statistical analysis were done by using the software GraphPad Insatat3

Preliminary Phytochemical Analysis
The tests were performed for the presence of different chemical constituents in the crude extracts of B. lycium roots and the results are shown as followings ( Table 2).

DISCUSSION
B. lycium is traditionally used for gastrointestinal disorders [3,6] therefore current study was conducted to validate its uses and underlying mechanisms involved. Bl.Aq exhibit a slight spasmogenic effect at lower concentration, followed by spasmolytic effect at higher concentration which indicates the presence of a combination of gut stimulant and inhibitory constituents in the aqueous extract.
In the presence of atropine; a muscarinic receptor antagonist [31] contractile effect was partially suppressed indicates the presence of cholinergic components in the aqueous extract of B. lycium. To see whether the contractile effect was mediated through an Ach-like mechanism, Bl.Aq was further tested on guinea pig ileum; a quiescent preparation [32] and the results were comparable with that of rabbit jejunum; indicates that Bl.Aq causes gut stimulation through multiple components including cholinergic. In GIT smooth muscles, M 3 receptors are present, which increase the intracellular calcium level, leads to increase the gastric motility and secretion [31] and the presence of cholinergic component(s) in the plant extract give the logical reasons for the use of B. lycium as carminative, stomachic and aperient.
K + at high concentration (>30mM) is known to cause smooth muscle contraction through opening of voltage-dependent L-type Ca 2+ Channels, thus allowing an influx of extracellular Ca 2+ into cytosol causing a contractile effect [33,34] and a substance causing inhibition of high K + induced contraction is considered as a blocker of Ca 2+ influx [35]. Since Bl.Aq inhibited high K + induced contractions, which may be due to calcium channel blocking activity. The purposed CCB effect was confirmed further by observing a dose-dependent rightward shift in the Ca ++ concentration response curve, similar to verapamil, a Ca ++ channel blocker [36]. Since control of GI smooth muscles is largely dependent on intracellular calcium concentration. While increased intercellular calcium stimulates the secretary processes and motility can leads to diarrhoea and the voltage gated calcium channels trigger this calcium influx [31,37]. So a calcium channel blocker can suppress GI motility, they are useful in hyperactive gut disorders like diarrhea and abdominal cramps [38] and the presence of calcium antagonist activity in plant extract may give the possible explanation of the use of plant in diarrhea and intestinal colic.
To evaluate its usefulness in condition associated with motility of the gut, Bl.Meth was also tested and the effects were almost comparable in all level of experiments performed for Bl.Aq, except with slight difference that spasmogenic effect was slightly depressed and spasmolytic effect was dominant in Bl.Meth as compared to Bl.Aq on rabbit jejunum and guinea pig ileum.

CONCLUSION
By taking in consideration the data, obtained from the experimental work it is strongly suggested that aqueous and methanolic extracts of B. lycium roots contain non-specific spasmogenic activity, partially mediated through cholinergic substances and further experimental work is required to find out the other components involved, while the spasmolytic effect is likely to be occurring through CCB like activity that supports its traditional uses in diarrhea, intestinal cramps and other gastrointestinal intestinal disorders. On the bases of these findings, it could be recommended that B. lycium plant can be used as safer medicine for GIT disorders in Human.

CONSENT
Not applicable.

ETHICAL APPROVAL
All authors hereby declare that "Principles of laboratory animal care" (NIH publication No. 85-23, revised 1985) were followed, as well as specific national laws where applicable. All experiments have been examined and approved by the appropriate ethics committee. All authors hereby declare that all experiments have been examined and approved by the ethical committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 declaration of Helsinki."