Spasmolytic and Antimicrobial Activities of Crude Extract of Bongardia chrysogonum L. Tubers

MATERIALS AND METHODS

Reagents: Drugs and analytical grade chemicals were purchased from Sigma (St. Louis, MO, USA). All drugs were dissolved in distilled water and dilutions were prepared fresh on the day of the experiment.

Animals: Male Wistar rats (250±8.11 g) were obtained from the Faculty of Medicine, University of Jordan, subjected to a 24 h fast and given access to water ad libitum prior to experimentation. All procedures involving animals were carried out in accordance with Jordanian regulations for animal experimentation and care. The protocol was reviewed and approved by the laboratory animal ethics committee of the Scientific Research Council at the Deanship of Academic Research (protocol and ethics approval number UJ 218/2013-2014). The study commenced on December 1, 2014 and lasted for 24 months. All experimentation was carried out at the pharmacology research lab in the Faculty of Medicine. The University of Jordan, Amman, Jordan. All efforts were made to minimize animal suffering and reduce the numbers of animal used.

Duodenum preparation: Duodenum preparation was carried out according to the method described by Shatarat et al.¹⁶. After pretreating the tissue with BC extract or control, the cumulative concentration response curve obtained using acetylcholine (ACh) chloride, potassium chloride (KCl) and barium chloride (BaCl₂) was recorded isotonically and the effect was allowed to reach a steady state at each concentration.

Plant materials and extraction: Bongardia chrysogonum tubers were collected inspiring in Jordan (15 km of Northwest Amman). The collected material was identified by Professor Suleiman Al-Olimat, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan. A voucher for each plant material has been deposited in the herbarium of the University for Future Reference (BC No. 5450).

Tubers were selected, dried completely under shade and then ground to a fine powder. Crude extracts were prepared by refluxing 500 g of the powdered plant material with 80% ethanol or chloroform for 3 h and maintaining the extract for 5 days at room temperature with continuous shaking (Labtech, Korea). The crude extracts were then passed through 125 mm filter paper (Albet, EEC). The volume of each filtered solution was increased to 100 mL with solvent and then the solvent was evaporated under reduced pressure using a rotary evaporator (Heidolph Laborota, Germany) to obtain 10% crude extracts (100 mg mL^–1). The extracts were further dried by incubating at room temperature for 8 days. The crude extracts were either used directly or stored in an air-tight container for further use.

Assessment of antispasmodic activity: After stabilization for 30 min, the antispasmodic effect of the BC tuber ethanol extract was evaluated by using the following procedures.

Relaxant effect of BC tuber extract on ACh-induced contractions: After immersion and equilibration of the duodenum in the organ bath, the dose-response curve was obtained for ACh. An increase in ACh concentration resulted in increased smooth muscle contraction and the submaximal contractile response was obtained at 3×10^–5 M. This concentration was selected for the cumulative concentration response curves. Increasing concentrations of BC ethanol extract were added to the organ bath 2 min before ACh administration and the effect of the BC extract was expressed as a percentage of the ACh-induced contraction.

Relaxant effect of BC tuber extract on BaCl₂-induced contractions: Freshly isolated duodenum was also exposed to 5 mM BaCl₂ to induce contractions. Increasing concentrations of BC ethanol extract were added to the organ bath 2 min before the addition of BaCl₂ and the effect of the BC extract was expressed as a percentage of the BaCl₂-induced contraction.

Relaxant effect of BC tuber extract on KCl-induced contractions: To determine Ca²⁺ antagonist activity, 60 mM KCl was added to the organ bath to produce sustained contractions. Increasing concentrations of BC ethanol extract were then added to evaluate its inhibitory effects, which were expressed as a percentage of the KCl-induced contractions.

Solvent effect on rat duodenum contractions: The effect of the solvent (80% ethanol) on smooth muscle contractions in the isolated rat duodenum was evaluated by adding up to 100 μL ethanol to the organ bath.

Antimicrobial activity: The agar well diffusion method was used to evaluate antimicrobial activity of the BC extracts¹⁷ against Bacillus subtilis (ATCC 11562), Staphylococcus aureus (ATCC 6538), Staphylococcus epidermidis (ATCC 12228), Escherichia coli (ATCC 29425) and Pseudomonas aeruginosa (ATCC 11921). Petri plates were filled with 75 mL seeded agar, which was allowed to solidify. Freshly prepared bacterial inoculum was evenly spread using a sterile cotton swab over the entire agar surface. A 6 mm hole was then punched with a sterile cork borer and 100 μL each of ethanolic and aqueous crude BC tuber extract was poured into the well. The Petri plates were allowed to stand at room temperature for 1 h and then incubated at 37°C overnight. The solvent dimethyl sulfoxide (DMSO) was used to fill the well as a negative control and 1 mg mL^–1 nor floxacin was used as a positive control. The zone of inhibition (average diameter) after 24 h was determined for each treatment and compared with the controls.

Determination of minimum inhibitory concentration and minimum bactericidal concentration: To confirm the results of the agar diffusion assay, the Minimum Inhibitory Concentration (MIC) of both BC extracts was determined by the microdilution method¹⁸ using nutrient broth. The MIC is defined as the lowest concentration of an antimicrobial that inhibits visible growth of a microorganism after overnight incubation. Plant extracts were dissolved in 10% DMSO and two fold dilutions were prepared with nutrient broth. The nutrient broth containing plant extract was inoculated with 10 μL bacterial suspension (5×10⁶ CFU mL^–1), with nutrient broth containing DMSO serving as a negative control. To evaluate bacterial growth, 270 mg resazurin was dissolved in 40 mL sterile water and 10 μL of the resazurin solution was added to each sample and incubated for 24 h at 37°C. Bacterial growth was detected by reading absorbance at 500 nm. Bacterial growth was indicated by a color change from purple to pink or colorless (assessed visually). The MIC was defined as the lowest plant extract concentration at which the color changed or the highest dilution that completely inhibited bacterial growth.

The samples were then spread on fresh solid media and incubated for 18 h to determine the Minimum Bactericidal Concentration (MBC) values (i.e., lowest concentration that prevents the growth of an organism). The lowest plant extract concentration that prevented all bacterial growth was reported as the MBC. The MIC and MBC were determined in triplicate experiments.

Preliminary phytochemical analysis: Qualitative phytochemical analysis of the ethanolic and chloroform BC tuber extracts was performed according to the standard procedures described by Harborne¹², Trease and Evans¹⁸ and Wagner et al.¹⁹. The analysis consisted of the foam test for saponins^12,19, Liebermann-Burchard test for terpenoids and triterpenoids¹⁸, FeCl₃ and lead subacetate tests for tannins^12,19, Shinoda’s test for flavonoids¹⁹, Borntrager’s test for anthraquinones^12,19 Keller-Killiani test for cardiac glycosides¹⁸, Molisch’s test for carbohydrates¹², Fehling’s test for reducing sugars¹², Barfoed’s test for monosaccharides¹², Millon’s test for proteins¹², ninhydrin test for amino acids¹² and Mayer’s test, Dragendorff’s test, Wagner’s test and 1% picric acid foralkaloids^12,19. The results are expressed as (+) for the presence and (-) for the absence of the phytochemical.

Statistical analysis: Results are expressed as Mean±Standard Error of the Mean (SEM) of 4-6 replicates, as indicated in the figure legends. Results were compared by one-way analysis of variance (ANOVA), followed by the post hoc Dunnett’s multiple comparison test¹⁹, p<0.05 was considered significant. Statistical analysis was performed using GraphPad Prism version 5.02 for Windows (GraphPad Software, San Diego, CA, USA).

RESULTS

Effect of BC ethanolic extract on spontaneous contraction in isolated rat duodenum: In all experiments, the addition of ACh, BaCl₂ (5 mM) or KCl (60 mM) caused a rapid contraction in the duodenum, which returned to the plateau state immediately. This contraction of duodenum (without BC tuber extract) was considered 100%.

Effect of BC tuber extract on ACh-induced contractions in isolated rat duodenum: A single application of 3×10^–5 M ACh evoked a contraction of 100% (Fig. 2), which was inhibited in a concentration-dependent manner by 0.15- 2.5 mg mL^–1 BC tuber extract. This relaxant effect was reversible and the spontaneous activity returned to normal after washing the preparation.

Effect of BC tuber extract on BaCl₂-induced contractions of isolated rat duodenum: The contractile response produced by a single application of BaCl₂ (5 mM) was considered 100% (n = 6). The results showed that BC tuber extracts (0.15-2.5 mg mL^–1) decreased the contractile response evoked by BaCl₂. No significant decrease was observed with 0.15 mg mL^–1 BC extract; however, contractions were 84.4% of the control (p<0.01) with 0.5 mg mL^–1 extract (Fig. 3).

Fig. 2:	Concentration-dependent inhibitory effects of crude BC tuber extract on spontaneous contractions and contractions induced by 3×10–5 M ACh in isolated rat duodenal smooth muscle
	Results are expressed as Mean±SEM (n = 4), *p<0.01, ***p<0.001 (ANOVA followed by Dunnett’s test, IC50= 1.25 mg mL–1)

Fig. 3:	Concentration-dependent inhibitory effects of crude BC tuber extract on spontaneous contractions and contractions induced by 5 mM BaCl2 in isolated rat duodenal smooth muscle
	Results are expressed as Mean±SEM (n = 6), **p<0.01, ***p<0.001 (ANOVA followed by Dunnett’s test, IC50 = 1.5 mg mL–1)

Effect of BC tuber extract on KCl-induced contractions in isolated rat duodenum: The addition of KCl (60 mM) caused sustained contractions (n = 4) in isolated rat duodenum (Fig. 4). The KCl-induced contractions were not significantly affected by 0.15-2 mg mL^–1 BC extract; however, 250 mg mL^–1 BC tuber extract decreased KCl-induced contractile responses to 53±6% that of the control.

Solvent effect on rat duodenum contraction: To determine whether the solvent used to prepare the BC tuber extract (70% v/v ethanol) could induce smooth muscle contraction, up to 100 μL ethanol was added to the organ bath.

Fig. 4:	Concentration-dependent inhibitory effects of crude BC tuber extract on spontaneous contractions and contractions induced by 60 mM KCl in isolated rat duodenal smooth muscle
	Results are expressed as Mean±SEM (n = 4), *p<0.01 (ANOVA followed by Dunnett’s test)

Fig. 5:	Effect of 70% ethanol on isolated rat duodenum compared with control (no treatment)
	Results are expressed as Mean±SEM (n = 4)

Spontaneous rhythmic contractions were not significantly affected by the addition of ethanol (Fig. 5).

Antimicrobial activity: The antimicrobial potential of BC was qualitatively evaluated by the presence or absence of inhibition zones and the inhibition zone diameter. The MIC and MBC were determined by using the agar well diffusion method, with norfloxacin serving as a positive control. Results summarized in Table 1 and 2 show that ethanolic BC tuber extract did not inhibit the growth of either Gram-positive or Gram-negative bacteria. Using ethanolic and aqueous tuber extracts, no antimicrobial activity was detected against B. subtilis, S. aureus, S. epidermidis, E. coli or P. aeruginosa, suggesting that the plant tuber lacks antimicrobial activity.

Table 1:	Antimicrobial activity recorded as zone of inhibition of Bongardia chrysogonum extracts against selected bacterial strains, using norfloxacin as a positive control
Results are expressed as Mean±SEM

Table 2:	Minimum Inhibitory Concentration (MIC) and Minimum Bacterial Concentration (MBC) of Bongardia chrysogonum extracts against selected bacterial strains, using norfloxacin as a positive control
Results are expressed as Mean±SEM

Table 3:	Qualitative phytochemical screening of Bongardia chrysogonum tubers
+++: Highly present, ++: Moderately present, +: Slightly present, -: Absence of bioactive compound

This is the first study reporting antimicrobial activity of this plant species against selected bacteria strains under these assay conditions.

Preliminary phytochemical analysis: Preliminary phytochemical screening of ethanolic and chloroform BC tuber extracts revealed the presence of the following secondary metabolites: Alkaloids, tannins, monosaccharides and other carbohydrates (Table 3). Saponins were detected only in the ethanolic extract. Analysis of the chloroform BC tuber extract showed relatively small levels of alkaloids and tannins, whereas the ethanolic extract showed relatively high levels of alkaloids, saponins and tannins.

DISCUSSION

Our results (Fig. 2-5) showed that BC tuber extract was able to not only relax the tone of spontaneous contractions in the isolated rat duodenum but also attenuated the spasmogenic effects of ACh, BaCl₂ and KCL at high concentrations. These spasmogens cause contractions by different mechanisms. The spasmolytic effect of BC tuber extract was reversible and the spontaneous activity returned to normal after washing the tissue three times. Furthermore, the effect of the ethanolic extract was concentration-dependent.

ACh, KCl and BaCl₂-induced contractions are commonly used to analyze the spasmolytic activity and/or mechanism of action of drugs and plant extracts in vitro. The effects of ACh are receptor-mediated, whereas the effects of KCl are Ca²⁺ channel-mediated and BaCl₂ is a nonspecific smooth muscle spasmogen^20-23. The rhythmic contractions of smooth muscle cells depend on an endogenous pacemaker driven by the cytosolic Ca²⁺ oscillator, which is responsible for the periodic release of Ca²⁺ from the endoplasmic reticulum. These pulses of Ca²⁺ often cause membrane depolarization²⁴. The study showed that BC extract inhibits ACh-induced contractions in a dose-dependent manner, suggesting an anticholinergic action. ACh-induced contractions in the rat ileum involve two different mechanisms involving muscarinic receptors²⁵. The ACh binds to M₂ and M₃ receptors in the gut tissue, causing smooth muscle contraction by promoting calcium influx via receptor-operated calcium channels²⁶. In addition, ACh promotes inositol triphosphate synthesis via phospholipase c activation, which in turn increases calcium release from the sarcoplasmic reticulum²⁴.The results showed that ACh alone causes contractions in isolated rat duodenum, and ethanolic BC tuber extract markedly decreased ACh-induced contractions. This result suggests that ethanolic BC tuber extract exerts strong spasmolytic activity by blocking cholinergic receptors, which may therefore be a potential target for BC.

High barium ion concentrations in the extracellular fluid depolarize the smooth muscle membrane and open voltage-dependent calcium channels, resulting in calcium in ux²³. The results of this study showed that BC tuber extract significantly p<0.05 inhibited BaCl₂-induced contractions. This inhibitory effect did not involve a decrease in smooth muscle myofilament sensitivity to Ca²⁺, because the effect was not reversed by washing. It is possible that BC tuber extract contains compounds that interfere with Ca²⁺ channel activity or with Ca²⁺ ion release from intracellular stores.

L-type Ca²⁺ channels have been identified in the rat intestine. High K⁺ stimulation provokes membrane depolarization and is the most common method used to introduce Ca²⁺ into the cell without receptor stimulation^22,23. It has been reported that high K⁺concentrations (>30 mM) open voltage-dependent calcium channels, causing Ca²⁺ influx and smooth muscle contraction²⁰. In this study, 60 mM K⁺ was used to cause depolarization of the tissue. The addition of BC tuber extract inhibited the K⁺-induced contractions only at the highest concentration tested (2.5 mg mL^–1).

BaCl₂ depolarizes the smooth muscle membrane and opens voltage-dependent Ca⁺ channels, resulting in Ca²⁺ influx. The BC tuber extract significantly p<0.05 inhibited BaCl₂-induced contractions. This relaxant effect of BC tuber extract on BaCl₂-induced contractions was concentration-dependent and greater than its effect on KCl-induced contractions. These results suggest that the antispasmodic activity of BC tuber extract may beat least partially mediated by targeting voltage-dependent Ca⁺ channels.

Phytochemical studies of the BC tuber demonstrated the presence of alkaloids^11-13 and saponins¹⁴ as the main secondary metabolites, with potential biological activities. Therefore, the spasmolytic activity of BC tuber extract observed in this study may be attributed in part to these metabolites. However, additional studies are needed to test this hypothesis. Antispasmodic activity could be attributed individual bioactive constituents or may be the result of a general inhibitory effect of multiple constituents that inhibit Ca²⁺ influx.

Preliminary phytochemical screening is useful to detect bioactive compounds and may lead to drug discovery and development. Results of these tests may also provide evidence for the traditional medical uses of B. Chrysogonum and claims about its antispasmodic effects. Future studies are needed to isolate, identify, characterize and elucidate the structure of bioactive compounds of BC.

No detectable antimicrobial activity against gram-positive and gram-negative bacteria was observed for crude BC tuber extract using the agar disk diffusion and microdilution assays. These negative results may be due to the nature of the phytochemical components of the plant and the bacteria used in the antimicrobial tests. The three major chemical constituents of pharmacological interest in the BC tuber extract were saponins, alkaloids and tannins, all of which contribute considerably to its biological activity. Previous studies report that saponins show only weak or no growth inhibition against both gram-positive and gram-negative bacteria (MIC>000 g mL^–1)^27,28. Alkaloids can have potent antimicrobial activities but this effect depends on their chemical nature, their structural class and the type of bacteria tested²⁹, which could explain the negative antimicrobial assay results. Tannins have potential as antibacterial compounds when used alone³⁰ but may be present in insufficient concentrations in the BC tuber extracts to exert antimicrobial effects. In addition, other phytochemicals present in this plant may alter this activity.

No previous studies have published results of phytochemical screening, anti microbial assays or antispasmodic tests of B. chrysogonum. Results of this preliminary study indicate the need for future research to elucidate the bioactive constituents of this plant with respect to the pharmacological activities studied here.

CONCLUSION AND FUTURE RECOMMENDATION

This study showed that BC tuber extract exhibits significant antispasmodic activity on isolated rat duodenum, providing a scientific basis for the folk medicinal use of B. chrysogonum in the treatment of gastrointestinal disorders. Although no antimicrobial activity of the plant extract was detected, phytochemical analysis and published data demonstrate the presence of alkaloids and saponins, which may account for the antispasmodic activity. The phytochemical compounds identified in this study may be responsible for the beneficial effects of B. chrysogonum. Extracts from this plant may therefore serve as a good source for useful drugs; however, future research is needed to isolate, purify and characterize the bioactive constituents responsible for the antispasmodic activity of B. chrysogonum.

SIGNIFICANCE STATEMENTS

This study investigated the antimicrobial properties and antispasmodic potential of Bongardia chrysogonum L. tuber extract in an isolated rat duodenum model, demonstrating that this extract may have beneficial effects as a novel spasmolytic. This study lends pharmacological credence to the folkloric, ethnomedical use of these plant tubers as a natural remedy for the treatment of abdominal cramps and various gastrointestinal tract disorders.

ACKNOWLEDGMENTS

This project was sponsored by the Deanship of Scientific Research at the University of Jordan (grant number SRF/JU/218/2013-2014). I would like to extend my thanks to the University of Jordan for giving me (main author) a sabbatical leave for the academic year 2016-2017.