Preliminary evaluation of the antispasmodic and lipoxygenase inhibitory effects of some selected medicinal plants

Abstract

The present study aimed to evaluate the possible antispasmodic and lipoxygenase inhibitory activity of some Pakistani medicinal plants. Extracts from Aconitum laeve Royle (Ranunculaceae), Trichodesma indicum Linn. (Boraginaceae), and Sauromatum guttatum Schott (Araceae) (corms) were tested on the isolated rabbit jejunum. All the extracts caused reduction in spontaneous and acetylcholine-induced contractions. A. laeve displayed excellent spasmolytic activity and almost (95%) diminished the normal contraction of rabbit jejunum at a concentration of 0.25 mg/mL of final bath. T. indicum inhibited the intestinal contractions by 78% at 5 mg/mL while S. guttatum (corms) caused 69% inhibition of spontaneous contractions at a concentration of 2 mg/mL. Extracts from A. laeve, T. indicum, S. guttatum (leaves and berries) and Paeonia emodi Wall. (Paeoniaceae) were screened in vitro for lipoxygenase inhibitory activity. All the extracts except A. laeve, showed good to excellent inhibition of the tested enzyme. P. emodi and S. guttatum (leaves) each inhibited the enzyme by 90% while T. indicum and S. guttatum (berries), respectively, showed 64.5% and 65% inhibitory activity against this enzyme.

Keywords::

• Antispasmodic
• lipoxygenase inhibition
• Aconitum laeve
• Trichodesma indicum
• Sauromatum guttatum
• Paeonia emodi

Introduction

Smooth muscle spasm plays an important role in conditions such as asthma, diarrhea, irritable bowel ­syndrome, kidney stones, and dysmenorrhea. Treatment of such conditions usually involves the relaxation of the smooth muscles in the walls of the affected organs (Mans et al., 2004). For example, some gastrointestinal disorders are currently treated by the inhibition of smooth muscle contractions (Sadraei et al., 2003). The search for new medicines with this property still presents an important field of research.

Arachidonate 5-lipoxygenase is the key enzyme that catalyzes the conversion of arachidonic acid to leukotrienes which are active mediators of inflammatory and allergic reactions (Ahmad et al., 2008; Khattak et al., 2005; Wagner, 1993). Lipoxygenase inhibition is thus a key target for the design and discovery of new therapies for the treatment of a variety of inflammatory diseases including bronchial asthma, cancer and autoimmune diseases (Ahmad et al., 2008; Khattak et al., 2005; Wagner, 1993).

Pakistan has a wealth of medicinal plants and thus is rich in herbal remedies. Moreover, the rural population is greatly dependent on the indigenous ­system of medicine for its health-related problems (Ahmad et al., 2007). Plants possess enormous ­potential to ­furnish new drugs for the treatment of various ailments. Different strategies are used to search for new bioactive principles from higher plants. One of the most reliable approaches is the random collection of ­readily available plants and their extraction followed by a broad screening for one or more types of pharmacological activity. This method eventually should result in the identification of potentially useful drugs (Farnsworth, 1988). We have studied several plants of Pakistan for various pharmacological and biological activities following this strategy. These include, among others, Trichodesma indicum Linn. (Boraginaceae), Aconitum laeve Royle (Ranunculaceae), Sauromatum guttatum Schott (Araceae) (Khan et al., 2006a, 2007), and Paeonia emodi Wall. (Paeoniaceae) (Khan et al., 2005a, 2005b; Khan & Ahmad, 2007). These plants are commonly available in Pakistan and find several applications in traditional systems of medicine (Khan et al., 2006b).

In our earlier studies on extracts of T. indicum, A. laeve and S. guttatum, agglutination of human RBCs and weak radical scavenging properties were observed (Khan et al., 2006a). S. guttatum also displayed antimicrobial activity (Khan et al., 2006a), and a good and moderate α-chymotrypsin inhibitory activity was observed for T. indicum and S. guttatum (leaf) extracts, respectively (Khan et al., 2006a). The S. guttatum extract exhibited significant toxicity against brine shrimp (Khan et al., 2006a). These plants also showed marked phytotoxic and insecticidal activities (Khan et al., 2007). Similarly, the aerial parts of P. emodi showed excellent enzyme (urease and α-chymotrypsin) inhibitory and radical scavenging activities (Khan et al., 2005a). The extract also displayed excellent phytotoxicity, moderate hemagglutination and insecticidal activity (Khan et al., 2005b) and excellent antispasmodic activity (Khan & Ahmad, 2007).

The current study was designed to extend this search for potential novel agents that may be of therapeutic application. Here, we report the screening of the plants T. indicum, A. laeve, S. guttatum, and P. emodi for antispasmodic and/or lipoxygenase inhibitory activities in order to rationalize some of their traditional uses or otherwise to explore the presence of these effects in the studied plants.

Materials and methods

Plant material and preparation of extract

The plants T. indicum (whole plant), A. laeve (roots) and P. emodi (aerial parts) were collected in July-August from the Swat, Pakistan. All these plants were identified by Mehboob-ur-Rehman, plant taxonomist, Department of Botany, Government Degree College Matta, Swat, Pakistan. The voucher specimens were deposited at the Herbarium Post-Graduate Jehanzeb College, Swat, Pakistan. S. guttatum (corms, leaves, and berries) plants were collected from Mohmund Agency, Pakistan in the month of July. The identification of the plant was confirmed by Abdur Rashid, Department of Botany, University of Peshawar, Peshawar, Pakistan. A voucher specimen was deposited at the Herbarium of the same department.

Air-dried and ground plant material of T. indicum (4.8 kg), S. guttatum (5.5 kg) and P. emodi (2.1 kg) were extracted with ethanol for three weeks. A. laeve (1 kg) was first defatted with n-hexane and then the residue was extracted with ethanol. All these extracts were filtered and subsequently evaporated under vacuum to dryness (T. indicum: 300 g; S. guttatum: 315 g; P. emodi: 355 g; A. laeve: 80 g) as described previously (Khan et al., 2005a, 2006a).

Antispasmodic activity

Antispasmodic activity of the extracts was ­evaluated using the isolated rabbit jejunum as reported previously (Khan & Ahmad, 2007). Rabbits used in this study were from local breeds of both sex weighing 1.2-1.7 kg and purchased from the local market. These animals were acclimatized for a reasonable time before being used in the study. The animals food was withdrawn 24 h prior to the experiment, but they had free excess to water during this fasting period. Briefly, the animals were ­sacrificed by stunning followed by exsanguination. Segments of jejunum were mounted in ­oxygenated Tyrode’s solution maintained at 37°C in an organ bath and allowed to equilibrate for 30 min. The crude extracts were dissolved in 2-3 mL purified water and added to the organ bath. All experiments were performed in triplicate (n = 3) and the results were expressed as mean ± SEM. The following formula was used for calculations:

Lipoxygenase inhibition assay

Lipoxygenase inhibitory activity was undertaken by slightly modifying the spectrophotometric method developed by Tappel (1962). Briefly, lipoxygenase (type I-B) and linoleic acid were purchased from Sigma (St Louis, MO). Aliquots of 160 μL 100 mM sodium phosphate buffer (pH 8), 10 μL crude extract solution (1 mg/mL extract in methanol) and 20 μL lipoxygenase solution were mixed and incubated for 10 min at 25°C. The reaction was then initiated by the addition of 10 μL linoleic acid solution (substrate), with the formation of (9Z,11E)-(13S)-13-hydroperoxyoctadeca-9,11-dienoate, the change in the reaction was followed for 10 min. Crude extract and the control were dissolved in methanol. All the reactions were performed in triplicate in 96-well plates. Percentage inhibitions were calculated from the following formula:

Results and discussion

Antispasmodic activities of the selected plants

The extracts obtained from the selected plants were investigated at various concentrations using isolated rabbit jejunum, and all displayed significant ­activity (Table 1). A. laeve exhibited a marked spasmolytic ­activity in a dose-dependent fashion and was found to be the most potent of the tested extracts. At a ­concentration of 0.01 mg/mL, it caused 11.77% inhibition of ­spontaneous contraction. Inhibition increased linearly with increasing concentration of the extract to a maximum effect of 95% inhibition of contractions at 0.25 mg/mL final bath ­concentration (Table 1). Alkaloids have been reported to be the principal class of constituents from A. laeve (Ulubelen et al., 2002; Shaheen et al., 2005). Indeed, phyto-chemical studies of this extract have led us to the isolation of several alkaloids; the exact structures of which are currently under investigation. Therefore, it is possible that the significant antispasmodic activity exhibited by this extract may be due to the presence of alkaloid(s).

Table 1.  Antispasmodic activity of crude extracts derived from selected plants of Pakistan on isolated rabbit’s jejunum. The concentration (mg/mL) represents the final bath concentrations.

Extract	Concentration (mg/mL)	Inhibition of Contractions (%)
A. laeve	0.01	11.77 ± 2.63
	0.03	31.55 ± 6.54
	0.05	38.31 ± 7.21
	0.1	57.01 ± 10.54
	0.25	95.07 ± 4.77
T. indicum	0.1	6.55 ± 1.78
	0.3	16.07 ± 5.72
	0.5	19.06 ± 4.7
	1	29.99 ± 11.44
	3	49.54 ± 16.09
	5	77.98 ± 2.97
S. guttatum (corms)	0.1	7.78 ± 3.13
	0.3	30.81 ± 5.23
	0.5	53.57 ± 13.71
	1	64.46 ± 6.41
	2	69.49 ± 2.96

The T. indicum extract displayed a weaker spasmolytic activity than that of A. laeve. Thus, only small inhibitions in the jejunum contractions were observed at concentrations below 1 mg/mL. However, it caused a 30% inhibition when the final bath concentration was raised to 1 mg/mL. With further increase in the concentration, the inhibitions of jejunum contractions rose to a significant level. At 3 and 5 mg/mL, the inhibition was 50% and 78%, respectively (Table 1). T. indicum is used in traditional medicine in several gastrointestinal disorders (Khan et al., 2006a, 2006b). The current study confirms the presence of antispasmodic constituent(s) in this plant and thus provides a scientific basis for its traditional medical uses.

S. guttatum (corms) displayed antispasmodic activity, inhibiting the spontaneous contractions of the ­isolated jejunum by 69% at the highest tested ­concentration (2 mg/­mL). At a level of 0.1 mg/mL, 8% inhibition of ­jejunum contractions occurred. Significant ­increments were seen in inhibition of ­contractions when the concentration level was increased to 0.3 and 0.5 mg/mL. The inhibitory activity at these concentrations was found to be 31% and 54%, respectively. However, with further increase in the final bath ­concentration of the extract, very little further increase in spasmolytic ­activity occurred. The inhibition observed at 1 and 2 mg/mL was 65% and 70%, respectively (Table 1). Although several phytolectins have been previously isolated from the corms of S. ­guttatum (Shangary et al., 1995), it has not been thoroughly studied for other constituents. We have recently isolated ­several ­alkaloids from this extract which are currently under investigation for their detailed structures.

As all the crude extracts from the selected plants showed spasmolytic activity, they were tested for their possible anticholinergic activity using acetylcholine-induced contractions of the isolated rabbit jejunum. The results are shown in Figure 1 which represents the tracings from the typical experiments. All the extracts reduced the acetylcholine-induced contractions of the isolated rabbit jejunum to various degrees. A. laeve extract was found to be the most potent in this regards.

Figure 1.  Tracings from the typical experiments showing the effects of extracts from selected plants on acetylcholine-induced spontaneous contractions using isolated ileum of rabbits, acetylcholine only (a); A. laeve (b); S. guttatum (corms) (c); T. indicum (d).

During the current investigation all extracts of the selected plants displayed spasmolytic activity which confirms the presence of antispasmodic constituents in these plants. This study thus provides a rational basis for the use of the plants in traditional systems of medicines for these properties. In addition, for the plants which are currently not used for such medical indications, this study provides indications for possible extension of their uses in controlling spasticities of various types.

Lipoxygenase inhibitory effects of the selected plants

The results of in vitro lipoxygenase inhibitory activity obtained with the crude extracts of the selected plants are shown in Figure 2. The extract derived from the aerial parts of P. emodi demonstrated excellent lipoxygenase inhibitory activities. The extract inhibited the tested enzyme by 90%. Similarly, the extract derived from leaves of S. guttatum also exhibited excellent enzyme inhibition potency against lipoxygenase and it also inhibited the enzyme by 90%. The extracts obtained from berries of S. guttatum showed good inhibitory activity against lipoxygenase and caused 65% inhibition of this enzyme (Figure 2). The extracts derived from T. indicum ­demonstrated good enzyme inhibitory activity against lipoxygenase and cause the inhibition of the enzyme by 64.5%. In the case of A. laeve, the weakest lipoxygenase inhibitory activity (37.7%) was observed of all selected plants (Figure 2).

Figure 2.  In vitro lipoxygenase inhibitory effects of the extracts from the selected medicinal plants.

The search for new lipoxygenase inhibitors appears to be a promising approach for the development of new drugs for the treatment of a variety of disorders including bronchial asthma, inflammation, cancer and autoimmune diseases (Ahmad et al., 2008; Khattak et al., 2005; Wagner, 1993). From the present study it is evident that the plant kingdom is a valuable source for new 5-lipoxygenase inhibitors. Plant screening opens the possibility of finding lead structures which could be candidates for chemical modifications with the aim of optimizing bioavailability and pharmacokinetics of the active agent, thus considerably improving the efficiency of plant constituents for therapy (Khattak et al., 2005; Pietta et al., 1998; Pulido et al., 2000). Overall, this investigation supports the rational use of these extracts in traditional medical practice. From the finding of 5-lipoxygenase activity, it appears that P. emodi (aerial parts) and S. ­guttatum (leaves) are appropriate for further investigations, for which the isolation and identification of the active constituent(s) are priority.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.